Tumor antigens and CTL clones isolated by a novel procedure

ABSTRACT

The present invention relates to nucleic acid molecules encoding antigenic peptides from MAGE molecules that bind to HLA. An example of the nucleic acid molecules of the present invention is a nucleic acid molecule coding for the peptide GVYDGREHTV (SEQ ID NO: 44), which peptide binds to HLA-A2. The nucleic acid molecules and the encoded antigenic peptides are useful for diagnosing and treating various pathological conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/IB99/01664, filed on Sep. 15, 1999,which is a continuation-in-part of U.S. Ser. No. 09/289,350, filed onApr. 9, 1999, now U.S. Pat. No. 6,531,451, which is acontinuation-in-part of U.S. Ser. No. 09/165,863, filed on Oct. 2, 1998,now U.S. Pat. No. 6,407,063.

FIELD OF INVENTION

The present invention relates to isolation of cytotoxic T lymphocyte(CTL) clones. The CTL clones of the present invention have been isolatedby successive steps of stimulation and testing of lymphocytes withantigen presenting cells which present antigens derived from differentexpression systems, e.g., from recombinant Yersinia, recombinantSalmonella, or recombinant viruses. The present invention furtherrelates to isolated CTL clones that are specific for proteins of theMAGE family. Antigenic peptides as well as the peptide/HLA complexeswhich are recognized by the isolated CTL clones are also provided.

BACKGROUND

An important facet of the immune response in a mammalian subject is therecognition by T cells of the complexes of the cell surface molecules,i.e., the complexes of peptides and HLA (human leukocyte antigens) orMHC (major histocompatibility complexes) molecules. These peptides arederived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecules. See in this regard, Male et al.,Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters6-10. The interaction between T cell and HLA/peptide complexes isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. This mechanism is involved in the immune systemresponse to foreign materials, in autoimmune pathologies, and inresponses to cellular abnormalities.

Most progressively growing neoplastic cells express potentiallyimmunogenic tumor-associated antigens (TAAs), also called tumorrejection antigens (TRAs). A number of genes have been identified thatencode tumor rejection antigen precursors (or TRAPs), which areprocessed into TRAs in tumor cells. Such TRAP-encoding genes includemembers of the MAGE family, the BAGE family, the DAGE/PRAME family, theGAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase,Melan-A/MART-1, gp 100, MUC-1, TAG-72, CA125, mutated proto-oncogenessuch as praise, mutated tumor suppressor genes such as p53, tumorassociated viral antigens such as HPV16 E7. See, e.g., review by Van denEynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9:684-693,Sahin et al. (1997) in Curr. Opin. Immunol. 9:709-716, and Shawler etal. (1997) Advances in Pharmacology 40: 309-337, Academic Press, Inc.,San Diego, Calif.

TRAs, like other antigenic epitopes, are presented at the surface oftumor cells by MHC molecules and have been shown to induce a CTLresponse in vivo and in vitro. See, for example, van der Bruggen et al.(1991) Science 254: 1643-1647. However, such TRA expressing tumor cellsdo not provoke reliable anti-tumor immune responses in vivo that arecapable of controlling the growth of malignant cells. Boon et al. (1992)Cancer Surveys 13: 23-37; T. Boon (1993) Int. J. Cancer 54: 177-180; T.Boon (1992) Advances Cancer Res. 58: 177-209. Thus, generation of CTLclones that recognize specific TRAs provides a powerful tool for tumortherapeutics. The identification of TRAs also allows the design ofrecombinant vaccines for the treatment of various pathologicalconditions.

The present invention provides a novel procedure for isolating CTLclones. By following such procedure, novel CTL clones have been isolatedthat recognize specific antigenic peptides of proteins, preferably ofthe MAGE family. The MHC molecules presenting these peptides have beenidentified as well.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides methods for isolatingCTL clones from a blood sample.

The methods of the present invention include successive steps ofstimulating and testing lymphocytes with antigen presenting cells. Suchmethods, by employing different antigen presenting cells at differentsteps, significantly reduce non-specific CTL activities generated in theprocedure and permit more efficient isolation of CTL clones.

Antigen presenting cells which are used in the methods of the presentinvention can differ in cell type and/or in the expression system fromwhich the antigen to be presented is derived. Cells which can beemployed as antigen presenting cells in the present methods includeprofessional and facultative antigen presenting cells. A preferredantigen presenting cell is an autologous dendritic cell, an autologous Bcell transformed with EBV, or an activated T cell.

Antigen presenting cells can be modified by a variety of ways to effectthe expression of an antigen of interest at the cell surface,preferably, by infection with a recombinant Yersinia, recombinantSalmonella, or recombinant viruses. Preferred recombinant virusesinclude vaccinia, canarypox virus, other pox viruses, adenovirus, herpessimplex virus, and retrovirus.

The protein against which CTL clones are generated can be a tumorassociated protein, an antigenic protein of a pathogen, or the like.Preferably, the protein is a member of the MAGE family, in particular,MAGE-A1, MAGE-A3 and MAGE-A4.

In another embodiment, the present invention contemplates CTL clonesisolated by using the methods of the present invention.

In a preferred embodiment, the present invention provides isolated CTLclones that are specific for peptide/HLA complexes SAYGEPRKL(SEQ ID NO:2)/HLA-Cw3, DPARYEFLW(SEQ ID NO: 42)/HLA-B53, GVYDGREHTV(SEQ ID NO:44)/HLA-A2, SAFPTTINF(SEQ ID NO: 47)/HLA-Cw2, EVYDGREHSA(SEQ ID NO:48)/HLA-A28, AELVHFLLL (SEQ ID NO: 55)/HLA-B40, and RVRFFFPSL (SEQ IDNO: 57)/HLA-B7, respectively.

In a more preferred embodiment, the present invention provides isolatedCTL clones LB1137 462/F3.2, LB1801 456/H7.11, LB1118 466/D3.31, LB 1801456/H8.33, LB1137 H4.13, LB1841 526/F7.1 and LB1803 483/G8.4.

Furthermore, the present invention provides methods of identifyingantigenic peptide epitopes of a protein by using CTL clones isolatedfollowing the methods of present invention.

In still another embodiment, the present invention provides newlyisolated antigenic peptides, DPARYEFLW (MAGE-A1 258-266) (SEQ ID NO:42), GVYDGREHTV (MAGE-A4 230-239) (SEQ ID NO: 44), SAFPTTINF(SEQ ID NO:47) (MAGE-A1 62-70), EVYDGREHSA(SEQ ID NO: 48) (MAGE-A1 222-231),AELVHFLLL (SEQ ID NO: 55) (MAGE-A3 114-122), RVRFFFPSL (SEQ ID NO: 57)(MAGE-A1 289-297). Nucleic acid sequences encoding such peptides arealso contemplated.

In another embodiment, the present invention provides isolatedpeptide/HLA complexes, peptide SAYGEPRKL (SEQ ID NO: 2) complexed withHLA-Cw3, peptide DPARYEFLW(SEQ ID NO: 42) complexed with HLA-B53,peptide GVYDGREHTV(SEQ ID NO: 44) complexed with HLA-A2, peptideSAFPTTINF(SEQ ID NO: 47) complexed with HLA-Cw2, EVYDGREHSA(SEQ ID NO:48) complexed with HLA-A28, AELVHFLLL (SEQ ID NO: 55) complexed withHLA-B40, and RVRFFFPSL (SEQ ID NO: 57) complexed with HLA-B7.

In another embodiment, cells expressing any of these peptide/HLAcomplexes are contemplated.

Still another embodiment of the invention provides pharmaceuticalcompositions which include any one of the isolated CTL clones, theantigenic peptides, the peptide/HLA complexes, and cells expressing thepeptide/HLA complexes of the present invention.

In a further aspect, the present invention provides methods useful fordiagnosing and treating various pathological conditions.

One embodiment of the present invention provides methods of diagnosingin a subject, a pathological condition characterized by an abnormalexpression of a peptide/HLA complex, by detecting the presence of cellsabnormally expressing such complex in the subject.

Another embodiment of the present invention provides methods ofdetecting in a subject, the presence of cells abnormally expressing apeptide/HLA complex of the present invention by using an isolated CTLclone of the present invention which specifically recognizes suchcomplex.

One embodiment of the present invention provides methods of diagnosingin a subject, a pathological condition characterized by an abnormalexpression of a peptide/HLA complex, by detecting an increased frequencyof CTL cells specific for such complex.

Another embodiment of the present invention provides methods ofdetecting in a subject the presence, of CTL cells specific for apeptide/HLA complex of the present invention by using an antigenpresenting cell expressing such complex at the cell surface.

In still another embodiment, the present invention provides methods oftreating a subject of a pathological condition characterized by anabnormal expression of a peptide/HLA complex of the present invention byadministering to the subject, a therapeutically effective amount ofcells of a CTL clone specific for such complex.

Another embodiment provides methods of treating a subject of apathological condition characterized by an abnormal expression of apeptide/HLA complex of the present invention, by administering to thesubject a therapeutically effective amount of the peptide.

Still another embodiment provides methods of treating a pathologicalcondition characterized by an abnormal expression of a peptide/HLAcomplex of the present invention, by obtaining antigen presenting cellsfrom the subject, modifying such cells to effect a presentation of thepeptide/HLA complex at the cell surface, and then reperfusing such“loaded” cells into the subject.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates the plasmid map of the expression vectorpMS111-MAGE-A1 (YopE₁₃₀-MAGE-A1). The sequences of the primers PVB127and PVB128 are set forth in SEQ ID NO: 33 and SEQ ID NO: 34,respectively.

FIGS. 2A-2B depicts the procedure for stimulating CTL 82/30 withEBV-transformed human B cells (HLA-A1) mixed with recombinant Yersinia;FIG. 2B depicts the quantitation of IFN-released by activated CTLs.

FIGS. 3A-D depict the specific recognition by CTL clone LB1137 462/H7.11of a MAGE-A1 antigenic peptide (SEQ ID NO: 2) presented by HLA-B53.

FIGS. 4A-C depict the specific recognition by CTL clone LB1801 456/H7.11of a MAGE-A1 antigenic peptide (SEQ ID NO: 42) presented by HLA-B53.

FIGS. 5A-D depict the specific recognition by CTL clone LB1137 H4.13 ofa MAGE-A4 antigenic peptide (SEQ ID NO: 44) presented by HLA-A2.

FIG. 6 depicts the MAGE-A4 nucleotide sequences (SEQ ID NO: 49) and theprimers used in PCR as described in Example 9.

FIGS. 7A-D depict a MAGE-A1 peptide presented by HLA-Cw2 to CTL cloneLB1118 466/D3.31.

7A. Lysis by CTL clone LB1118 466/D3.31 of autologous EBV-B cellsinfected with vaccinia-MAGE-A1. Target cells were infected for 2 hoursat an MOI of 20, ⁵¹Cr-labeled, and incubated with CTL clone LB1118466/D3.31 for 4 hours. Targets infected with the parental vaccinia wereused as a negative control.

7B. Stimulation of CTL clone LB1118 466/D3.31 by COS-7 cells that weretransiently transfected with a MAGE-A1 cDNA and a cDNA encoding HLA-Cw2.One day after transfection, 1,500 CTL clone LB1118 466/D3.31 were addedinto microwells containing 1.5×10⁴ transfected COS-7 cells. TNFproduction was estimated after overnight coculture by testing thetoxicity of the supernatants for the TNF-sensitive cells of WEHI-164clone 13.

7C. Lysis by CTL clone LB1118 466/D3.31 of autologous EBV-B cellsincubated with synthetic peptide SAFPTTINF(SEQ ID NO: 47)(MAGE-A1₆₂₋₇₀). Targets were ⁵²Cr-labeled and incubated for 4 hours withthe CTL, at an effector-to-target ratio of 5:1, in the presence of thepeptide at the concentrations indicated.

7D. Lysis of HLA-Cw2 tumor cell lines by CTL clone LB1118 466/D3.31.Target cells were ⁵¹Cr-labeled and incubated for 4 hours with CTL cloneLB1118 466/D3.31 at various effector-to-target ratios.

FIGS. 8A-D depict a MAGE-A1 peptide presented by HLA-A28 to CTL cloneLB1801 456/H8.33.

8A. Lysis by CTL clone LB1801 456/H8.33 of autologous EBV-B cellsinfected with vaccinia-MAGE-A1. Target cells were infected for 2 hoursat an MOI of 20, ⁵¹Cr labeled, and incubated with CTL clone LB1801456/H8.33 for 4 hours. Targets infected with the parental vaccinia wereused as a negative control.

8B. Stimulation of CTL clone LB1801 456/H8.33 by COS-7 cells transientlytransfected with a MAGE-A1 cDNA and a cDNA encoding HLA-A28. One dayafter transfection, 1,500 CTL clone LB1801 456/H8.33 were added intomicrowells containing 1.5×10⁴ transfected COS-7 cells. TNF productionwas estimated after overnight coculture by testing the toxicity of thesupernatants for the TNF-sensitive cells of WEHI-164 clone 13.

8C. Lysis by CTL clone LB1801 456/H8.33 of autologous EBV-B cellsincubated with synthetic peptide EVYDGREHSA(SEQ ID NO: 48)(MAGE-A1₂₂₂₋₂₃₁). Targets were ⁵¹Cr-labeled and incubated for 4 hourswith CTL clone LB1801 456/H8.33, at an effector-to-target ratio of 5:1,in the presence of the peptide a the concentrations indicated.

8D. Lysis of HLA-A28 melanoma line by CTL clone LB1801 456/H8.33. Targetcells were ⁵¹Cr-labeled and incubated for 4 hours with CTL clone LB1801456/H8.33 at various effector-to-target ratios.

FIGS. 9A-D. A MAGE-A3 peptide is presented to CTL clone LB1841 526/F7.1by HLA-B40.

9A. Lysis by CTL clone LB1841 526/F7.1 of autologous EBV-B cellsinfected with vaccinia-MAGE-A3.

9B. Stimulation of CTL clone LB1841 526/F7.1 by COS-7 cells transientlytransfected with a MAGE-A3 cDNA and a cDNA encoding an HLA molecule asindicated.

9C. Lysis by CTL clone LB1841 526/F7.1 of autologous EBV-B cellsincubated with synthetic peptide AELVHFLLL (SEQ ID NO: 55)(MAGE-A3₁₁₄₋₁₂₂).

9D. Lysis of HLA-B40 melanoma cells by CTL clone LB1841 526/F7.1.

FIGS. 10A-D. A MAGE-A1 peptide is presented to CTL clone LB1803 483/G8.4by HLA-B7.

10A. Lysis by CTL clone LB1803 483/G8.4 of autologous EBV-B cellsinfected with vaccinia-MAGE-A1.

10B. Stimulation of CTL clone LB1803 483/G8.4 by COS cells transientlytransfected with a MAGE-A1 cDNA and a cDNA encoding an HLA molecule asindicated.

10C. Lysis by CTL clone LB1803 483/G8.4 of autologous EBV-B cellsincubated with synthetic peptide RVRFFFPSL (SEQ ID NO: 57)(MAGE-A1₂₈₉₋₂₉₇).

10D. Lysis of HLA-B7 melanoma cells by CTL clone LB1803 483/G8.4.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention provides novel methods forisolating CTL clones. The present methods include successive steps ofstimulating and testing lymphocytes by using different antigenpresenting cells at different steps.

The procedure to develop specific CTL clones in vitro has beendescribed. Briefly, a blood sample containing T-cell precursors is takenfrom a mammal. PBLs are purified from such blood sample and areincubated with stimulator cells which express antigenic peptidescompleted with the appropriate MHC molecule. Stimulator cells can betumor cells (see, e.g., the U.S. Pat. No. 5,342,774, Knuth et al. (Proc.Natl. Acad. Sci. USA 86: 2804-2808, 1989) and Van Den Eynde et al. (Int.J. Cancer 44: 634-640, 1989), or antigen presenting cells pulsed withdefined peptides. Additional components, e.g., allogeneic feeder cellsand cytokines, can be added into the incubation mixture. CTLs specificfor antigens expressed at the surface of the stimulator cells willproliferate, and thus, will be enriched in the cell population as aresult of the stimulation. CTL clones can be subsequently isolated by,e.g., limiting dilution. However, the approach using antigen presentingcells pulsed with defined peptides as stimulator cells, have sometimesgenerated CTLs that are unable to recognize the relevant tumor cells.

The present inventors have found that efficient isolation of CTL clonescan be achieved by successive steps of stimulating and testing T cellprecursors, using different antigen presenting cells at different steps.The present methods of isolating CTL clones permit significant reductionof CTL activities generated toward non-specific molecules, e.g.,molecules expressed from the backbone sequence of an expression vector.

By “different antigen presenting cells” it means that the antigenpresenting cells may differ in cell type or in the expression systemfrom which an antigen of interest being presented is derived.

“Antigen presenting cells” as referred herein, express at least oneclass I or class II MHC determinant and may include those cells whichare known as professional antigen-presenting cells such as macrophages,dendritic cells and B cells. Other professional antigen-presenting cellsinclude monocytes, marginal zone Kupffer cells, microglia, Langerhans'cells, interdigitating dendritic cells, follicular dendritic cells, andT cells. Facultative antigen-presenting cells can also be used accordingto the present invention. Examples of facultative antigen-presentingcells include activated T cells, astrocytes, follicular cells,endothelium and fibroblasts. As used herein, “antigen-presenting cells”encompass both professional and facultative types of antigen-presentingcells.

The antigen presenting cells can be isolated from tissue or bloodsamples (containing peripheral blood mononuclear cells) obtained from amammal such as human. Cell lines established from such samples may alsobe used. Procedures for establishing cell lines are well known in theart. Certain cell lines may be obtained directly from the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209. Both normal and malignant cells can be employed.

Preferably, the MHC determinants expressed by the antigen presentingcells are compatible with those expressed by the mammal from which thesample containing T cell precursors is taken. More preferably,autologous antigen presenting cells or cell lines established therefromare employed. Non-autologous cells may be used as long as the MHCdeterminants expressed by such cells are compatible, either naturally,by way of transfection or other means that are appropriate. One skilledin the art is also familiar with the methods for determining whether theMHC molecules expressed by an antigen presenting cell are compatiblewith those of the mammal subject involved, such as well known HLA-typingprocedures. See general teachings by Coligan et al. (1994) CurrentProtocols in Immunology John Wiley & Sons Inc: New York, N.Y.

Preferred antigen presenting cells are autologous dendritic cells,autologous B cells transformed with EBV, and autologous T cell activatedby PHA.

Further, according to the present invention, antigen presenting cellsused in the present methods can also differ in the expression systemfrom which an antigen of interest is derived. More specifically, theantigen presenting cells can be modified in various ways to effect theexpression of an antigen at the cell surface. For example, an antigenpresenting cell can be infected with a recombinant Yersinia, arecombinant Salmonella, or a recombinant virus. In each case, therecombinant microorganism encodes a protein from which the peptideantigen presented is derived.

The protein expressed from any of these expression systems is processedin the antigen presenting cells into small peptides, which are thencomplexed with the appropriate MHC molecules and presented at the cellsurface. In the present invention, peptides that are complexed with MHCmolecules and presented at the cell surface are also referred to as“antigens”.

The term “Yersinia” as used herein includes all species of Yersinia,including Yersinia enterocolitica, Yersinia pseudotuberculosis andYersinia pestis. The term “recombinant Yersinia” used herein refers toYersinia genetically transformed with an expression vector. The term“delivery” used herein refers to the transportation of a protein from aYersinia to an antigen presenting cell, including the steps ofexpressing the protein in the Yersinia, secreting the expressedprotein(s) from such Yersinia and translocating the secreted protein(s)by such Yersinia into the cytosol of the antigen presenting cell.

According to the present invention, preferred Yersinia for use inexpressing and delivering the protein of interest are mutant Yersiniathat are deficient in producing functional effector proteins.

A preferred mutant Yersinia strain for use in expressing and deliveringthe protein of interest is a quintuple-mutant Yersinia strain in whichall the effector-encoding genes are mutated such that the resultingYersinia no longer produce any functional effector proteins. Suchquintuple-mutant Yersinia strain is designated as yopEHOMP for Y.enterocolitica or yopEHAMJ for Y. pseudotuberculosis. One example ofsuch yopEHOMP strain is Y. enterocolitica MRS40(pABL403).

An antigenic protein of interest can be cloned into a yersiniaexpression vector Ffr used in combination with a mutant Yersinia fordelivery of the protein into antigen presenting cells. In accordancewith the present invention, such a vector is characterized by (in the 5′to 3′ direction) a promoter, a first nucleic acid sequence encoding adelivery signal, a second nucleic acid sequence fused thereto coding forthe protein to be delivered and other sequences that may be appropriate(e.g., a polyadenylation signal).

The promoter of the expression vector is preferably from a Yersiniavirulon gene. A “Yersinia virulon gene” refers to genes on the YersiniapYV plasmid, the expression of which is controlled both by temperatureand by contact with a target cell. See review by Cornelis et al. (1997).Such genes include genes coding for elements of the secretion machinery(the Ysc genes), genes coding for translocators (YopB, YopD, and LcrV),genes coding for the control elements (YopN and LcrG), and genes codingfor effectors (YopE, YopH, YopO/YpkA, YopM and YopP/YopJ). Preferably,the promoter is from an effector-encoding gene selected from any one ofYopE, YopH, YopO/YpkA, YopM and YopP/YopJ. More preferably, the promoteris from YopE.

Further, in accordance with the present invention, a first DNA sequencecoding for a delivery signal is operably linked to the promoter. “Adelivery signal”, as described hereinabove, refers to a polypeptidewhich can be recognized by the secretion and translocation system ofYersinia and therefore directs the secretion and translocation of aprotein into a an antigen presenting cell. Such polypeptide is from aneffector protein including YopE, YpPH, YopO/YpkA, YopM, and YopP/YopJ,and preferably, YopE. More preferably, the effector protein is YopE ofYersinia enterocolitica.

One skilled in the art is familiar with the methods for identifying thepolypeptide sequences of an effector protein that are capable ofdelivering a protein. For example, one such method is described by Soryet al. (1994). Examples of such delivery signal polypeptides includefrom Y. enterocolitica: YopE₁₃₀ (the N-terminal 130 amino acids ofYopE), YopE₅₀, YopM₁₀₀ and YopH₇₁.

The yersinia expression vectors may be transformed into Yersinia by anumber of known methods which include, but are not limited to,electroporation, calcium phosphate mediated transformation, conjugation,or combinations thereof. For example, a vector can be transformed into afirst bacteria strain by a standard electroporation procedure.Subsequently, such a vector can be transferred from the first bacteriastrain into Yersinia by conjugation, a process also called“mobilization”. Yersinia transformant (i.e., Yersinia having taken upthe vector) may be selected, e.g., with antibiotics. These techniquesare well known in the art. See, for example, Sory et al. (1994).

The delivery of a protein from a recombinant Yersinia into the cytosolof an antigen presenting cell can be effected by contacting an antigenpresenting cell with a recombinant Yersinia under appropriateconditions. Multiple references and techniques are available for thoseskilled in the art regarding the conditions for inducing the expressionand translocation of virulon genes, including the desired temperature,Ca++ concentration, manners in which Yersinia and target cells aremixed, and the like. See, for example, Cornelis, Cross talk betweenYersinia and eukaryotic cells, Society for General MicrobiologySymposium, 55; Mocrae, Saunders, Smyth, Stow (eds), Molecular aspects ofhost-pathogen interactions, Cambridge University Press, 1997. Theconditions may vary depending on the type of eukaryotic cells to betargeted, e.g.: the conditions for targeting human epithelial carcinomaHela cells (Sory et al. (1994)); the conditions for targeting mousethymoma or melanoma cells (Starnbach et al. (1994) J. Immunol. 153:1603); and the conditions for targeting mouse macrophages (Boland et al.(1996)). Such variations can be addressed by those skilled in the artusing conventional techniques.

Those skilled in the art can also use a number of assays to determinewhether the delivery of a fusion protein is successful. For example, thefusion protein may be labeled with an isotope or an immunofluoresceine,or detected by a immunofluoresceine conjugated antibody, as disclosed byRosqvist et al. (1994) EMBO J. 13: 964. The determination can also bebased on the enzymatic activity of the protein being delivered, e.g.,the assay described by Sory et al. (1994). The determination can also bebased on the antigenicity of the protein being delivered. For example,the delivery of a MAGE-A1 protein into EBV-transformed human B cells canbe detected by the recognition of such targeted B cells by CTL cellsspecific for MAGE-A1 epitopes. Such CTL recognition, in turn, may bedetected by a number of assays including assaying the secretion of IFN-γfrom the activated CTLs or Cr⁵¹ release from lysed target cells. Methodssuch as Western-blot analysis using antibodies specific against theprotein being delivered, PCR in situ hybridization, or ELISPOT (MabtechAB, Sweden) may also be employed for such determination. See, e.g., W.Herr et al. (1997) J. Immunol. Methods 203: 141-152 and W. Herr et al.(1996) J. Immunol. Methods 191: 131-142.

In accordance with the present invention, the antigenic protein ofinterest can also be expressed from a recombinant Salmonella. Forexample, avirulent strains of Salmonella typhimurium can be used asantigen delivery vectors. It is known in the art that antigenicepitopes, such as viral epitopes can be successfully delivered to thehost cell cytosol by using the type III protein secretion system of S.typhimurium. See, e.g., Russmann et al. (1998) 281: 565-568.

In accordance with the present invention, the expression of a protein ofinterest in the antigen presenting cell can also be effected byinfection of the antigen presenting cells with a recombinant virus. Inparticular, the present invention contemplates recombinant viruses ofvaccinia, canarypox virus, other pox viruses, adenovirus, herpes simplexvirus, retrovirus and any other viruses that are appropriate.

A preferred strain of vaccinia for use in the present invention is theWR strain (Panicali et al. (1981), J. Virol. 37: 1000-1010). Thenucleotide sequence coding for the protein of interest can be operablylinked to a promoter, such as an vaccinia promoter H6, and inserted intoa vaccinia vector, thereby generating a donor plasmid. Vaccinia vectorswhich can be employed for generating adeno-plasmids are available tothose skilled in the art and are described in, e.g., U.S. Pat. No.4,769,330. Recombinant WR strains of vaccinia can be generated by usinga donor plasmid via in vivo recombination, following well-knownprocedures. See, e.g., Perkins et al., J. Virol. 63: 3829-3936 (1989).

A preferred strain of canarypox virus for use in the present inventionis ALVAC (Cox et al. (1993), Virology 195: 845-850). The nucleotidesequence coding for the protein of interest can be operably linked to apromoter, such as an vaccinia promoter H6, and inserted into an ALVACvector to create a donor plasmid. Multiple ALVAC vectors are availableto one skilled in the art and are described by, e.g., U.S. Pat. No.5,756,106; Cox et al. (1993) Virology 195: 845-850; Tartaglia et al.(1993) J. Virology 67: 2370-2375; and Taylor et al. (1992) Virology 187:321-328. Such donor plasmid can be used to generate recombinant ALVACviruses via in vivo recombination. See, e.g., Cox. et al. (1993);Tartaglia et al. (1993) and Taylor et al. (1992).

Those skilled in the art can also generate recombinant adenoviruses forexpressing the protein of interest as described in, e.g., Example 5hereinafter.

A nucleotide sequence encoding the antigenic protein of interest can becloned into the various expression vectors as described above. There isno particular limitation in the protein that can be employed in theinstant methods for isolating CTL clones.

The term “protein” as used herein refers to naturally occurring proteinsas well as artificially engineered proteins, or parts thereof. The term“part of a protein” includes a peptide fragment of a protein that is ofsufficient length to be antigenic. Preferably, such a fragment consistsof at least 8 or 9 amino acids. “Artificially engineered proteins” asused herein refer to non-naturally occurring proteins, e.g., modifiedforms of non-naturally occurring proteins, or fusion of two or morenaturally occurring proteins or parts thereof, which are also referredto as polytopes (in-frame fusion of two or more epitopes) as exemplifiedby Thompson et al. (1995) in Proc. Natl. Acad. Sci. USA 92: 5845-5849.

The present invention contemplates, in particular, tumor associatedproteins or pathogen associated antigens.

A “tumor associated protein” refers to a protein that is specificallyexpressed in tumors or expressed at an abnormal level in tumors relativeto normal tissues. Such tumor associated proteins include, but are notlimited to, members of the MAGE family, the BAGE family (such asBAGE-1), the DAGE/PRAME family (such as DAGE-1), the GAGE family, theRAGE family (such as RAGE-1), the SMAGE family, NAG, Tyrosinase,Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, mutated proto-oncogenessuch as p21ras, mutated tumor suppressor genes such as p53, tumorassociated viral antigens (e.g., HPV16 E7), the SSX family, HOM-MEL-55,NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11,HOM-MEL-2.4, HOM-TES-11, RCC-3.1.3, NY-ESO-1, and the SCP family.Members of the MAGE family include, but are not limited to, MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A11. Members of the GAGE family include,but are not limited to, GAGE-1, GAGE-6. See, e.g., review by Van denEynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9: 684-693,Sahin et al. (1997) in Curr. Opin. Immunol. 9: 709-716, and Shawler etal. (1997). These proteins have been shown to associate with certaintumors such as melanoma, lung cancer, prostate cancer, breast cancer,renal cancer and others.

A number of known antigenic proteins from pathogens are alsocontemplated by the present invention.

The pathogens can include viruses, bacteria, parasites and fungi.Specific examples of antigenic proteins characteristic of a pathogeninclude the influenza virus nucleoprotein (residues 218-226, as setforth in F. et al. (1997) J. Virol. 71: 2715-2721) antigens from Sendaivirus and lymphocytic choriomeningitis virus (see, An et al. (1997) J.Virol. 71: 2292-2302), the B1 protein of hepatitis C virus (Bruna-Romeroet al. (1997) Hepatology 25: 470-477), the virus envelope glycoproteingp 160 of HIV (Achour et al. (1996) J. Virol. 70: 6741-6750), aminoacids 252-260 or the circumsporozite protein of Plasmodium berghei(Allsopp et al. (1996) Eur. J. Immunol. 26: 1951-1958), the influenza Avirus nucleoprotein (residues 366-374, Nomura et al. (1996) J. Immunol.Methods 193: 4149), the listeriolysin O protein of Listeriamonocytogenes (residues 91-99, An et al. (1996) Infect. Immun. 64:1685-1693), the E6 protein (residues 131-140, Gao et al. (1995) J.Immunol. 155: 5519-5526) and E7 protein (residues 21-28 and 48-55, Baueret al. (1995) Scand. J. Immunol. 42: 317-323) of human papillomavirustype 16, the M2 protein of respiratory syncytial virus (residues 82-90and 81-95, Hsu et al. (1995) Immunology 85: 347-350), the herpes simplexvirus type 1 ribonucleotide reductase (see, Salvucci et al. 1995) J.Gen. Virol. 69: 1122-1131) and the rotavirus VP7 protein (see, Franco etal. (1993) J. Gen. Virol. 74: 2579-2586), P. falciparum antigens(causing malaria) and hepatitis B surface antigen (Gilbert et al. (1997)Nature Biotech. 15: 1280-1283).

A number of short antigenic peptides can also be employed in the presentinvention. One skilled in the art can readily determine the length ofthe fragments required to produce immunogenic peptides. Alternatively,the skilled artisan can also use coding sequences for peptides that areknown to elicit specific T cell responses (either CD4⁺ or CD8⁺ T cells),such as tumor-associated antigenic peptides (TAA, also known as TRAs fortumor rejection antigens) as disclosed by U.S. Pat. Nos. 5,462,871,5,558,995, 5,554,724, 5,585,461, 5,591,430, 5,554,506, 5,487,974,5,530,096, 5,519,117. Examples of TRAs are provided in Table 1. See alsoreview by Van den Eynde and van der Bruggen (1997) and Shawler et al.(1997). Antigenic peptides of a pathogen origin can also be used, suchas those disclosed by Gilbert et al. (1997).

TABLE 1 Exemplary Antigens SEQ ID Gene MHC Peptide Position NO: MAGE-HLA-A1 EADPTGHSY 161-169 1 A1 HLA-Cw16 SAYGEPRKL 230-238 2 MAGE- HLA-A1EVDPIGHLY 168-176 3 A3 HLA-A2 FLWGPRALV 271-279 4 HLA-B44 MEVDPIGHLY167-176 5 BAGE HLA-Cw16 AARAVFLAL  2-10 6 GAGE-1,2 HLA-Cw16 YRPRPRRY 9-16 7 RAGE HLA-B7 SPSSNRIRNT 11-20 8 GnT-V HLA-A2 VLPDVFIRC(V)   2-10/11 9 MUM-1 HLA-B44 EEKLIVVLF exon 2/ 10 intron EEKLSVVLF (wild11 type) CDK4 HLA-A2 ACDPHSGHFV 23-32 12 ARDPHSGHFV 13 (wild type)β-catenin HLA-A24 SYLDSGIHF 29-37 14 SYLDSGIHS (wild 15 type) TyrosinaseHLA-A2 MLLAVLYCL 1-9 16 HLA-A2 YMNGTMSQV 369-377 17 HLA-A2 YMDGTMSQV369-377 18 HLA-A24 AFLPWHRLF 206-214 19 HLA-B44 SEIWRDIDF 192-200 20HLA-B44 YEIWRDIDF 192-200 21 HLA-DR4 QNILLSNAPLGPQFP 56-70 22 HLA-DR4DYSYLQDSDPDSFQD 448-462 23 Melan- HLA-A2 (E)AAGIGILTV 26/27-35   24A^(MART-1) HLA-A2 ILTVILGVL 32-40 25 gp- HLA-A2 KTWGQYWQV 154-162 26100^(Pme1117) HLA-A2 ITDQVPFSV 209-217 27 HLA-A2 YLEPGPVTA 280-288 28HLA-A2 LLDGTATLRL 457-466 29 HLA-A2 VLYRYGSFSV 476-485 30 DAGE HLA-A24LYVDSLFFL 301-309 31 MAGE- HLA-Cw16 KISGGPRISYPL 292-303 32 A6

As described herein above, sequences coding for a full-length naturallyoccurring protein, a part of a naturally occurring protein, combinationsof parts of a naturally occurring protein, or combinations of differentnaturally occurring proteins or parts from different proteins, may allbe employed to be cloned into the expression vectors as describedhereinabove.

The present invention further provides recombinant expression vectorswhich can be employed in the present methods, including recombinantyersinia expression vectors, e.g., pMS111-YopE₁₃₀-MAGE-A1 andPMS111-YopE₁₃₀-MAGE-A4; recombinant vaccinia vectors, e.g., WR-MAGE-A1and WR-MAGE-A4; recombinant canarypox viral vectors, e.g.,ALVAC-MAGE-A1; recombinant adenoviral vectors, e.g., adeno-MAGE-A4; andretroviral vectors, e.g., M1-CSM.

To carry out the methods of the present invention, a sample containingT-cell precursors is obtained from a subject, typically, a blood samplefrom a human subject. The subject can be a cancer patient or anindividual without cancer. The sample may be treated to concentrateT-cell precursors prior to stimulation.

The sample is contacted with a first antigen presenting cell expressinga protein, along with any other materials that may be appropriate, suchas lymphokines. Upon contact, specific T-cell precursors are activatedand begin to proliferate.

Cells in the sample are subsequently tested by contacting the cells witha second antigen presenting cell expressing the protein. The sample canbe first diluted and distributed into microwells such that individualcells can be separately tested. CTL Cells which are specific for theprotein, or “responding CTLs”, can be identified and selected by avariety of standard assays such as a ⁵¹Cr release assay, a IFN-γsecretion assay, or a TNF production assay.

In a preferred embodiment of the present invention, the CTL cells thusselected are subject to at least one additional cycle of stimulation andtesting steps.

According to the present invention, the antigen presenting cells used atone step can differ from the cells used in a subsequent step, either incell type or in the expression system from which the protein isexpressed.

For testing the specificity of CTL responses after stimulation, antigenpresenting cells of a type that expresses high amounts of class I HLAmolecules are preferred, e.g., EBV-transformed B cells.

In a preferred embodiment of the present invention, one of theexpression systems used by the antigen presenting cell at one step(either stimulation or testing), is different from at least one of theother expression systems used in another step.

More Preferably, the antigen presenting cells used at a stimulation stepemploy an expression system different from that used in the immediatelyfollowing testing step.

The present invention provides examples of combinations of differentantigen presenting cells which can be used for isolating specific CTLclones. According to the present invention, CD8⁺ T lymphocytes obtainedfrom an individual can be stimulated in microwells with autologousmonocyte-derived dendritic cells infected with a recombinant ALVACcanarypoxvirus encoding a protein of interest. After several times ofstimulation, an aliquot of each microculture can then be tested forspecific lysis of autologous EBV-B cells infected with a recombinantVaccinia encoding the protein of interest. The positive microculturescan then be diluted and stimulated again with autologous EBV-B cellsinfected with a recombinant Yersinia encoding the protein of interest.Specific clones can be detected and thus isolated by testing forspecific lysis of autologous EBV-B cells infected with a recombinantVaccinia encoding the protein of interest. Thus, the combination ofantigen presenting cells used in the foregoing procedure can becharacterized asdendritic-ALVAC/EBV-B-Vaccinia/EBV-B-Yersinia/EBV-B-Vaccinia. Additionalpreferred combinations of antigen presenting cells which can be used inthe present methods include:dendritic-Adeno/EBV-B-Vaccinia/EBV-B-Yersinia/EBV-B-Vaccinia,dendritic-ALVAC/EBV-B-Vaccinia/T cell-retroviral/EBV-B-Vaccinia. Thepresent invention is not limited to the above exemplified combinations.

In a further aspect of the invention, the present invention contemplatesCTL clones isolated by using the methods of the present invention.

In another embodiment, the present invention contemplates methods foridentifying antigenic peptide epitopes of a protein. According to suchmethod, CTL clones that recognize certain antigenic epitopes of aprotein are isolated using the present method of isolating CTL clones,as described hereinabove. Such clones can then be used to identify thespecific antigenic peptides as well as the presenting HLA molecules,using a variety of well-known procedures, for example, proceduresdescribed in Examples 7-11.

According to the methods of the present invention, the identification ofan antigenic peptide epitope of a protein is based on the capacity ofthe peptide/HLA complex, at the surface of an antigen presenting cell,to activate the specific CTLs. The antigenic peptide epitopes thusidentified likely represent the epitopes that are well processed andadequately expressed at the cell surface in vivo. By using such methodof the present invention, antigenic peptide epitopes from proteins ofthe MAGE family have been identified; namely, MAGE-A1 peptide 230-238(presented by HLA-Cw3 and recognized by clone LB1137 462/F3.2), MAGE-A1peptide 258-266 (presented by HLA-B53 and recognized by clone LB1801456/H7.11), MAGE-A1 peptide 62-70 (presented by HLA-Cw2 and recognizedby clone LB 1118 466/D3.31), MAGE-A1 peptide 222-231 (presented byHLA-A28 and recognized by clone LB1801 456/H8.33), MAGE-A1 peptide289-297 (presented by HLA-B7 and recognized by clone LB1803 483/G8.4), aMAGE-A3 peptide 114-122 (presented by HLA-B40 and recognized by cloneLB1841 526/F7.1), and a MAGE-A4 peptide 230-239 (presented by HLA-A2 andrecognized by clone LB1137 H4.13). See Table 2. Among these, MAGE-A1peptide 230-238 (SAYGEPRKL (SEQ ID NO: 2)) has been previouslyidentified, but was found therein to be presented by a different HLAmolecule, HLA-Cw16 (U.S. Pat. No. 5,558,995).

TABLE 2 SEQ GENE POSITION PEPTIDE MHC CTL ID MAGE-A1 230-238 SAYGEPRKLHLA- LB1137 2 Cw3 462/F3.2 MAGE-A1 258-266 DPARYEFLW HLA- LB1801 42 B53456/H7.11 MAGE-A4 230-239 GVYDGREHTV HLA- LB1137 44 A2 H4.13 MAGE-A162-70 SAFPTTINF HLA- LB1118 47 Cw2 466/D3.31 MAGE-A1 222-231 EVYDGREHSAHLA- LB1801 48 A28 456/H8.33 MAGE-A3 114-122 AELVHFLLL HLA- LB1841 55B40 526/F7.1 MAGE-A1 289-297 RVRFFFPSL HLA- LB1803 57 B7 483/G8.4

Accordingly, another embodiment of the present invention providesisolated CTL clones that are specific for peptide/HLA complexesSAYGEPRKL(SEQ ID NO: 2)/HLA-Cw3, DPARYEFLW(SEQ ID NO: 42)/HLA-B53,GVYDGREHTV(SEQ ID NO: 44)/HLA-A2, SAFPTTINF(SEQ ID NO: 47)/HLA-Cw2,EVYDGREHSA(SEQ ID NO: 48)/HLA-A28, AELVHFLLL(SEQ ID NO: 55)/HLA-B40, andRVRFFFPSL(SEQ ID NO: 57)/HLA-B7, respectively.

In a preferred embodiment, the present invention provides isolated CTLclones LB1137 462/F3.2, LB1801 456/H7.11, LB1118 466/D3.31, LB1801456/H8.33, LB1137 H4.13, LB1841 526/F7.1 and LB1803 483/G8.4.

In another embodiment, the present invention is directed to the newlyisolated antigenic peptides, namely, DPARYEFLW(SEQ ID NO: 42) (MAGE-A1258-266), GVYDGREHTV(SEQ ID NO: 44) (MAGE-A4 230-239), SAFPTTINF(SEQ IDNO: 47) (MAGE-A1 62-70), EVYDGREHSA(SEQ ID NO: 48) (MAGE-A1 222-231),AELVHFLLL (SEQ ID NO: 55) (MAGE-A3 114-122) and RVRFFFPSL (SEQ ID NO:57) (MAGE-A1 289-297). Nucleic acid sequences encoding these peptidesare also contemplated.

Another embodiment of the present invention is directed to the isolatedpeptide/HLA complexes of the present invention. Specifically, thepresent invention provides isolated complex of peptide SAYGEPRKL(SEQ IDNO: 2) and HLA-Cw3, complex of peptide DPARYEFLW(SEQ ID NO: 42) andHLA-B53, complex of peptide GVYDGREHTV(SEQ ID NO: 44) and HLA-A2,complex of peptide SAFPTTINF(SEQ ID NO: 47) and HLA-Cw2, complex ofpeptide EVYDGREHSA(SEQ ID NO: 48) and HLA-A28, complex of AELVHFLLL (SEQID NO: 55) and HLA-B40, and complex of RVRFFFPSL (SEQ ID NO: 57) andHLA-B7.

Once the presenting HLA molecule for an antigenic peptide epitope hasbeen ascertained, a complex of the peptide and the HLA molecule can bemade by a variety of methods. For example, the HLA molecule can beproduced and isolated by any appropriate recombinant expression system,e.g., an E. coli-based expression system. Peptides can be made by, e.g.,chemical synthesis or recombinant expression. The peptides and the HLAmolecules can then be mixed in vitro under conditions that favor theformation of the HLA/peptide complexes. Such conditions are well knownin the art. See, e.g., Garboczi et al. (Proc. Natl. Acad. Sci. USA 89:3429-3433, 1992 and Altman et al. (Science 274: 94-96, 1996).

The present invention further contemplates cells expressing any of theinstant peptide/HLA complexes at the cell surface. Such cells can bemade using any antigen presenting cells that are appropriate includingcell lines (e.g., COS cells, CHO cells and the like), and by, e.g.,peptide loading, or cotransfection as described in the Examples of thepresent disclosure.

In another embodiment, the present invention contemplates pharmaceuticalcompositions which include any one of the isolated CTL clones, theisolated antigenic peptides, the isolated peptide/HLA complexes, theantigen presenting cells expressing peptide/HLA complexes of the presentinvention, or combinations thereof.

The pharmaceutical compositions of the present invention can includeother substances such as cytokines, adjuvants and pharmaceuticallyacceptable carriers. As used herein, a therapeutically acceptablecarrier includes any and all solvents, including water, dispersionmedia, culture from cell media, isotonic agents and the like that arenon-toxic to the host. Preferably, it is an aqueous isotonic bufferedsolution with a pH of around 7.0. The use of such media and agents intherapeutic compositions is well known in the art. Supplementary activeingredients can also be incorporated into the compositions.

In a further aspect of the present invention, the isolated CTL clones,the isolated antigenic peptides, the cells expressing the peptide/HLAcomplexes of the present invention are employed in various methods fordiagnosing a pathological condition in a subject, preferably, a humansubject.

The pathological conditions contemplated by the present inventioninclude tumors and infections by pathogens such as bacteria, parasites,fungus or virus, and the like.

The term “abnormal expression” as used herein refers to an expressionthat is not present in normal cells or an expression that is present innormal cells at a significantly lower level. In the present invention,“an abnormal expression” can also be used to refer to an unusualprocessing of a protein which gives rise to an antigenic epitope that isnot presented at the surface of normal cells.

In one embodiment, the present invention provides methods of diagnosingin a subject, a pathological condition characterized by an abnormalexpression a peptide/HLA complex, by detecting in the subject, thepresence of cells abnormally expressing such complex.

In another embodiment, the present invention provides methods ofdetecting in a subject the presence of cells abnormally expressing apeptide/HLA complex of the present invention, by using an isolated CTLclone specific for such complex.

According to the present invention, a sample containing the cellssuspected to be abnormal is obtained from the subject by, e.g., tissuebiopsy. The sample is then contacted with a CTL clone of the presentinvention. The presence of the abnormal cells can be determined bymeasuring the activity of the CTL clone (i.e., CTL response) usingstandard assays such as ⁵¹Cr release, IFN-gamma secretion, or TNFproduction.

In another embodiment, the present invention provides methods fordetecting in a subject, the presence of CTL cells specific for anisolated peptide/HLA complex of the present invention. Morespecifically, a blood sample is secured from the subject and contactedwith cells expressing the specific peptide/HLA complexes. The presenceof CTL cells specific for the complex can be detected by any of theapproaches described hereinabove, e.g., the lysis of the cellsexpressing the specific peptide/HLA complexes measurable by a standard⁵¹Cr release assay.

Furthermore, the frequency of CTLs specific for a peptide/HLA complexcan be assessed by, e.g., limiting dilution analysis or tetramer assays.By comparing with a normal individual, an increased frequency of CTLsspecific for a peptide/HLA complex in an individual, is indicative of apathological condition characterized by an abnormal expression of thecomplex. Accordingly, the present invention contemplates methods ofdiagnosing a pathological condition characterized by an abnormalexpression of a peptide/HLA complex by detecting an increased frequencyof CTLs specific for such peptide/HLA complex.

In a further aspect of the present invention, the isolated CTL clones,the isolated antigenic peptides, the cells expressing the peptide/HLAcomplexes of the present invention are employed in various methods fortreating a pathological condition in a subject, preferably, a humansubject.

The term “treating” is used to refer to alleviating or inhibiting apathological condition, e.g., inhibiting tumor growth or metastasis,reducing the size of a tumor, or diminishing symptoms of a pathogeninfection, by e.g., eliciting an immune response.

In one embodiment, an isolated CTL clone of the present invention can beadministered, in a therapy regimen of adoptive transfer, to a subjectsuffering a pathological condition characterized by an abnormalexpression of the peptide/HLA complex that is specifically recognized bysuch CTL clone. See teachings by Greenberg (1986) J. Immunol. 136 (5):1917; Riddel et al. (1992) Science 257: 238; Lynch et al. (1991) Eur. J.Immunol. 21: 1403; and Kast et al. (1989) Cell 59: 603 for adoptivetransfer. CTLs, by lysing the cells abnormally expressing such antigens,can alleviate or treat the pathological condition at issue, such as atumor and an infection with a parasite or a virus.

In another embodiment, the present invention provides methods oftreating a subject suffering a pathological condition characterized byan abnormal expression of a peptide/HLA complex, by administering theisolated peptides, or the peptide/HLA complexes, to the subject. Thepathological condition can be alleviated by, e.g., specific immuneresponses elicited due to the administered peptides or peptide/HLAcomplexes.

In another embodiment of the present invention, a subject suffering apathological condition characterized by an abnormal expression of apeptide/HLA complex of the present invention, can be treated byobtaining antigen presenting cells from the subject, modifying suchcells to effect a presentation of the peptide/HLA complex at the cellsurface, and then reperfusing such “loaded” cells into the subject. Themodification can be achieved by transfecting the isolated antigenpresenting cells with any appropriate expression vectors encoding thepeptide or the full-length protein, or by loading the cells with thepeptides at issue following a peptide loading procedure as described by,e.g., Nestle et al. (Nature Medicine 4: 328-332, 1998).

For treatment purposes, the isolated CTL clones, the peptides or thepeptide/HLA complexes, or the cells expressing the peptide/HLAcomplexes, can be administered to a subject alone or in combination withother appropriate materials, such as cytokines, adjuvants or apharmaceutical carriers. The amount of the CTL cells, the peptides, thepeptide/HLA complexes, or cells expressing the complexes, can bedetermined according the condition of the subject.

For additional teachings of diagnostic and therapeutic uses of isolatedCTLs and peptide/HLA complexes, see, e.g., Thomson et al. (1995) PNAS92: 5845; Altman et al. (1996) Science 274: 94-96; Dunbar et al. (1998)Current Biology 8: 413-416; Greenberg et al. (1986) J. Immunol. 136:1917; and Kast et al. (1989) Cell 59: 603-614.

The present invention is further illustrated by the following examples.

All the publications mentioned in the present disclosure areincorporated herein by reference. The terms and expressions which havebeen employed in the present disclosure are used as terms of descriptionand not of limitation, and there is no intention in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof, recognizing that variousmodifications are possible within the scope of the invention.

EXAMPLE 1 Generation of Recombinant Yersinia and TargetingEBV-Transformed B Cells with Recombinant Yersinia

Strains, Plasmids and Growth Conditions

Y. enterocolitica strain E40(pYV40), MRS40(pYV40), which is theisogeneic ampicillin sensitive derivative of E40(pYV40), and theirvarious non-polar mutants (Sory et al. (1995), Proc. Natl Acad. Sci. USA92: 11998-12002). Plasmids are listed in Table 3. Bacteria were grown inBrain Heart Infusion (BHI) (Difco, Detroit, Mich.). After overnightpreculture, bacteria were diluted {fraction (1/20)} in fresh BHI,allowed to grow for 30 minutes at room temperature, and synthesis of theYop virulon was induced by incubation for 150 minutes at 37° C. beforeinfection.

Construction of the Polymutant Yersinia Strains

To construct the yopHOPEM polymutant strain, the yopE, yopH, yopo, yopMand yopP genes were successively knocked out by allelic exchange in theMRS40 strain using the suicide vectors pMRS101 and pKNG101. See, K.Kaniga et al. (1991) “A wide-host range suicide vector for improvingreverse genetics in gram-negative bacteria: inactivation of the blaAgene of Yersinia enterocolitica” Gene 109: 137-141 and M. R. Sarker etal. (1997) “An improved version of suicide vector pKNG101 for genereplacement in Gram-negative bacteria” Mol. Microbiol. 23: 409-411. Thevarious deletions are described in Table 2 in the “suicide vectors andmutators” section. The YopE gene was first mutated using the mutatorpPW52 (see, P. Wattiau et al. (1993) “SycE, a chaperone-like protein ofYersinia enterocolitica involved in the secretion of YopE” Mol.Microbiol. 8: 123-131), giving strain MRS40(pAB4052). Mutation of theYopH gene in this strain with the mutator pAB31 (see, S. D. Mills et al.(1997) “Yersinia enterocolitica induces apoptosis in macrophages by aprocess requiring functional type III secretion and translocationmechanisms and involving YopP, presumably acting as an effector protein”Proc. Natl. Acad. Sci. USA 94: 12638-12643) gave the double yopEH mutantMRS40(pAB404). The triple yopEHO mutant MRS40(pAB405) was then obtainedby allelic exchange with the mutator pAB34 (see, S. D. Mills et al.,1997). The YopP gene was then mutated with mutator pMSK7 (see S. D.Mills et al. (1997)), leading to the yopEHOP mutant MRS40(pMSK46). TheyopHOPEM strain MRS40(pABL403) was finally obtained by allelic exchangewith the yopM mutator pAB38 (see, S. D. Mills et al., 1997).

TABLE 3 Plasmids Plasmids Relevant Characteristics References pABL403pYV40 yopE₂₁, yopHΔ¹⁻³⁵² see Example 2 of the yopOΔ⁶⁵⁻⁵⁵⁸, yopP₂₃,yopM₂₃ present specification Suicide Vectors and mutators pKNG101^(ori)R6K ^(aac)BR+ ^(on)TRK2 ^(str)AB+ K. Kaniga et al. (1991) Gene109: 137-141. pMRS101 ^(ori)R6K ^(sac)BR+ ^(on)TRK2 ^(str)AB+ M.R.Sarker and G.R. ^(ori)ColE1 bla + Cornelis (1997) Mol. Microbiol. 23:409-411. pAB31 pMRS101 yopHΔ₁₋₃₅₂+ S.D. Mills et al. (1997) Proc. Natl.Acad. Sci. USA 94: 12638-12643. pAB34 pMRS101 yopOΔ₆₅₋₅₅₈+ S.D. Mills etal. (1997) pAB38 pMRS101 yopM₂₃+ S.D. Mills et al. (1997) pMSK7 pMRS101yopP₂₃+ S.D. Mills et al. (1997) pPW52 pKNG101 yopE₂₁+ P. Wattiau andG.R. Cornelis (1993) Mol. Microbiol. 8: 123-131.

Generation of recombinant Yersinia containing YopE₁₃₀-MAGE-A1

The sequence encoding protein MAGE-A1 was inserted in frame with asequence encoding a truncated YopE, YopE₁₃₀, containing the first 130amino acids of YopE. Such a plasmid is graphically depicted in FIG. 1.

The open reading frame of MAGE-A1 was amplified by PCR using a MAGE-A1cDNA cloned in pcDNAI/Amp (Invitrogen, Carlsbad, Calif.) as template.The upstream primer, AAACTGCAGATGTCTCTTGAGCAGAGGAGTC (SEQ ID NO: 33),consisted of the first nucleotides of the open reading frame of MAGE-A1preceded by a PstI site. The downstream primer,AAACTGCAGTCAGACTCCCTCTTCCTCCTC (SEQ ID NO: 34), consisted of nucleotidescomplementary to the last nucleotides of the open reading frame ofMAGE-A1 followed by a PstI site. The PCR product was digested with PstIand inserted in frame with the truncated YopE at the PstI site of vectorpMS111 (see, Sory et al. (1994) Molecular Microbiology 14: 583-594), toyield plasmid YopE₁₃₀-MAGE-A1 or pMS111-MAGE-A1.

pMS111-MAGE-A1 (YopE₁₃₀-MAGE-A1) was electroporated in bacteria strainDH5 F′IQ. DNA was extracted from some clones and the DNA of a positiverecombinant clone was electroporated in bacteria strain SM10. Aftermobilization of pMS111 from SM10 in Yersinia MRS40 (pABL403),recombinant clones were then selected on agar-containing medium,supplemented with nalidixic acid, sodium-arsenite and chloramphenicol.MRS40 is an isogeneic derivative of E40 sensitive to ampicillin (see,Sory et al. (1995) Proc. Natl. Acad. Sci. USA 92: 11998-12002).

Generation of recombinant Yersinia containing YopE₁₃₀-MAGE-A4

The sequence encoding protein MAGE-A4 was linked in frame to a sequenceencoding a truncated YopE, YopE (1-130), containing the first 130 aminoacids of YopE. The open reading frame of MAGE-A4 was amplified by PCRusing a MAGE-A4 cDNA cloned in pcDNAI/Amp (Invitrogen) as template. Theupstream primer, AAAAACTGCAGATGTCTTCTGAGCAGAAGAGT (SEQ ID NO: 35),consisted of the first nucleotides of the open reading frame of MAGE-A4preceded by a PstI site. The downstream primer,AAAAAATCGATTCAGACTCCCTCTTCCTC (SEQ ID NO: 36), consisted of nucleotidescomplementary to the last nucleotides of the open reading frame ofMAGE-A4 followed by a ClaI site. The PCR was performed for 30 cycles (1min at 94° C., 2 min at 55° C. and 2 min at 72° C.). The PCR product wasdigested with PstI and ClaI and inserted in frame with the truncatedYopE at the PstI-ClaI sites of vector pMS621. Plasmid pMS621-MAGE-A4 wastransformed into bacteria strain DH5αF′IQ by electroporation. Positiveclones were detected by PCR on bacterial colonies and the DNA of apositive recombinant clone was extracted and transformed into bacteriastrain SM10 by electroporation. After mobilization of pMS621-MAGE-A4from SM10 into polymutant Yersinia MRS40 (pABL403), recombinant cloneswere then selected on agar-containing medium, supplemented withnalidixic acid, sodium m-arsenite and chloramphenicol.

Generation of recombinant Yersinia-MAGE-A3

Yersinia-MAGE-A3(aa147-314)

The sequences encoding the truncated MAGE-A3 was amplified by PCR usinga MAGE-A3 cDNA cloned in pcDNAI/Amp as template. The upstream primerPVB157 was 5′-AA CTGCAG TTTCCTGTGATCTTCAGCAAAGC-3′(SEQ ID NO: 50),consisting of nucleotides 439-461 of the open reading frame of MAGE-A3(start codon ATG is 1-3), preceded by a PstI site (in bold) and 2 A's.The downstream primer PVB139, 5′-CC ATCGAT TCACTCTTCCCCCTCTCTCAA-3′(SEQID NO: 51), consisting of nucleotides complementary to the lastnucleotides of the open reading frame of MAGE-A3 preceded by a ClaI site(in bold) and 2C's. PCR was performed for 30 cycles (1 min at 94° C., 2min at 62° C. and 2 min at 72° C.). The PCR products were treated asdescribed for MAGE-A4 hereinabove.

Construction of yersinia-MAGE-A3(aa1-199)

To obtain the sequence encoding the first 199 amino-acids of the MAGE-A3protein, the primers were designed as follows: upstream primer PVB172,5′-ACCAGAGTCATC CTGCAG ATGCCTCTTGAG -3′ (SEQ ID NO: 52), consisting ofthe nucleotides overlapping the start codon (underlined) of the openreading frame of MAGE-A3 preceded by a PstI site (in bold). Thedownstream primer PVB 173, 5′-GCCTGCCTTGGG ATCGAT TCACATGATCTGATT-3′(SEQID NO: 53), consisting of nucleotides complementary to the nucleotides577-600 of the open reading frame of MAGE-A3 and containing a ClaI site(in bold). PCR was performed for 30 cycles (1 min at 94° C., 2 min at65° C. and 2 min at 72° C.). The PCR products were treated as describedhereinabove for MAGE-A4.

Targeting EBV-Transformed B Cells with Recombinant Yersinia

Infection of EBV-transformed B cells with Yersinia MRS40 (pABL403)containing pMS111-MAGE-A1 was carried out as follows. Infection ofEBV-transformed B cells with other recombinant Yersinia was carried outfollowing essentially the same procedure.

One colony of Yersinia MRS40 (pABL403) containing pMS111-MAGE-A1 wasthen grown overnight at 28° C. in LB medium supplemented with nalidixicacid (35 μg/ml), sodium m-arsenite (1 mM) and chloramphenicol (12μg/ml). The overnight culture was diluted in fresh medium in order toobtain an OD (optical density) of 0.2 at 600 nm after amplifying thefresh culture at 28° C. for approximately 2 hours. The bacteria werewashed in 0.9% NaCl and resuspended at 10⁸ bacteria per ml in 0.9% NaClassuming that a culture giving an OD₆₀₀ equal to 1 contains 5×10⁸bacteria per ml. Irradiated EBV-B cells (100 Gy) were resuspended at 10⁶in 3.8 ml of RPMI without antibiotics, supplemented with 10% FCS and AAG(L-Arginine (116 mg/ml), L-Asparagine (36 mg/ml) and L-Glutamine (216mg/ml)). Then 200 μl of the bacterial suspension was added. Two hoursafter infection, gentamicin (30 μg/ml) was added for the nest two hours,and the cells were finally washed three times before being used asstimulator cells.

As a negative control, the same cells were also infected with YersiniaMRS40 (pABL403) containing pMS621, a plasmid which encodes only thetruncated YopE, i.e., YopE₁₃₀,

EBV-B Cells infected with the recombinant Yersinia-MAGE-A1 wererecognized by MZ2-CTL 82/30. MZ2-CTL 82/30 are specific for the MAGE-A1peptide EADPTGHSY (SEQ ID NO: 1) which is presented by HLA-A1 (U.S. Pat.No. 5,342,774). 5000 MZ2-CTL 82/30 cells were added in each microwellcontaining the Yersinia in a final volume of 100 μl of Iscove's completemedium (culture medium was supplemented with 10% human serum, L-arginine(116 mg/ml), L-asparagine (36 mg/ml), L-glutamine (216 mg/ml),streptomycine (0.1 mg/ml), penicillin (200 U/ml), IL-2 (25 U/ml) andgentamicin (15 μg/ml). After overnight incubation, the presence ofIFN-gamma (that is produced by CTL upon activation) in the supernatantof the co-culture was tested in a standard ELISA assay (Biosource,Fleurus, Belgium). FIG. 2A graphically depicts such a procedure.

As indicated in FIG. 2B, the HLA-A1⁺ B cells infected with Yersiniaencoding YopE₁₃₀-MAGE-A1 were recognized by the CTL 82/30, while thesame cells infected with the control plasmid YopE₁₃₀ were not. Theoptimal concentration of bacteria is around 1,000,000 per microwell.

EXAMPLE 2 Generation of Recombinant Vaccinia WR Viruses

Parental WR strain of Vaccinia (vP1170) contained the parent vectorpKILGPT of 2826 bp (Virogenetics, Troy, N.Y.). A sequence coding forMAGE-A1, placed after the Vacciniavirus H6 promoter, was cloned into thepKILGPT vector, creating donor plasmid MAW035. A similar MAGE-A4 donorplasmid vector was constructed by replacing the MAGE-A1 cDNA with theMAGE-A4 cDNA. The MAGE-A3 cDNA was digested with Pst1 and XbaI, bluntended, the insert was gel purified and ligated into the SmaI site of thePsC11 vector. For the PsC11 vector, see Chakrabati et al. (1985) Mol.Cell Biol. 5: 34-3-3409.

The donor plasmids was transfected into CEF cells containing the genomicDNA of vaccinia strain WR, yielding recombinant vaccinia virusesWR-MAGE-A1, WR-MAGE-A4 and WR-MAGE-A3, respectively, by way of in vivorecombination and selected with BrdU and X-gal. The procedure can befound in, e.g., Perkins et al. (1989) J. Virol. 63: 3829-3936.

EXAMPLE 3 Generation of Recombinant Alvac-Mage-A1 Viruses

A MAGE-A1 coding sequence, placed after the Vaccinia Virus H6 promoter,was cloned into the pUC8-based vector to generate donor plasmid MAW036.

Recombinant ALVAC-MAGE-A1 virus was generated by using the donor plasmidMAW036 and following well known procedures, e.g., as described inCurrent Protocols in Molecular Cloning (Ausubel et al., John Wiley &Sons, New York) and Ferrari et al. (Blood 90: 2406-2416, 1997).

Recombinant canarypox virus ALVAC-MAGE-A3 split (also referred to hereinas “ALVAC-MAGE-A3” for simplicity) expressed two truncated overlappingfragments of MAGE-A3, one fragment spanning amino acids 1-196 and theother fragment spanning amino acids 147-199. In the recombinant virus,each fragment was contained in a separate expression cassette, eachunder the control of the vaccinia virus H6 promoter, and both cassetteswere inserted at the C3 site in the ALVAC genome.

The vCP1563 recombinant was generated as follows. The MAGE-A3 (1-196)DNA fragment was generated and linked to the vaccinia H6 promoter bystandard PCR procedures with plasmid pTZ18R (containing full lengthMAGE-A3 cDNA) as template. The MAGE-A3 (147-199) DNA fragment wasgenerated and linked to the H6 promoter in the same way. These twofragments were then subcloned into a plasmid such that the cassetteswere flanked by ALVAC DNA from the C3 insertion site. The organizationof these elements in the plasmid was as follows: ALVAC C3 left flankingarm, MAGE-A3 (147-299)/H6, MAGE-A3 (1-196)/H6, ALVAC C3 right flankingarm. This ALVAC C3 site donor plasmid containing the MAGE-A3 (1-196) and(147-299) fragment expression cassettes was designated pC3MAGE3 split.

The ALVAC-MAGE-A3 recombinant was generated by in vivo recombinationbetween the pC3MAGE3 split donor plasmid and ALVAC genomic DNA followingstandard procedures. Recombinant virus was selected by hybridizationwith MAGE-3-specific DNA probes and plaque was purified. The resultingALVAC-MAGE-A3 recombinant was given the laboratory designation vCP1563.Expression analysis with MAGE-3-specific antisera confirmed theexpression of MAGE-A3 (1-196) and (147-299) polypeptides in cellsinfected with ALVAC-MAGE-A3 (vCP1563).

EXAMPLE 4 Generation of Recombinant Adenoviruses

For the construction of the recombinant adenovirus, the plasmidpAd-CMVIcpA-MAGE-A4 (containing the MAGE-A4 cDNA under the control ofthe CMV promoter) was obtained by inserting into the NotI site of vectorpAd-CMVIcpA (provided by Celia GARCIA and Thierry RAGOT, URA CNRS 1301),the MAGE-A4 complete cDNA.

The recombinant adenovirus Ad-MAGE-A4 was generated by in vivohomologous recombination in cell line 293 between pAd-CMVIcpA-MAGE-A4and βd-ggal genomic DNA. Briefly, 293 cells were cotransfected with 5 μgof plasmid pAd-CMVIcpA-MAGE-A4 linearized with XmnI and 5 μg of thelarge ClaI fragment of Adeno-βgal DNA (Stratford-Perricaudet et al.(1992), J. Clin. Invest., 90: 626-630 and Patent FR 9603207. Therecombinant adenovirus was plaque purified and the presence of thetransgene was assessed by restriction analysis of the adenoviral DNA.Recombinant adenoviruses were propagated in 293 cells and purified bydouble cesium chloride density centrifugation. The viral stocks werestored in aliquots with 10% glycerol in liquid nitrogen and titered byplaque assay using 293 cells.

Recombinant adenovirus Ad-MAGE-A3 was generated according to essentiallythe same procedure described above, but MAGE-A3 cDNA was derived from aλgt10 recombinant clone.

EXAMPLE 5 Recombinant Retrovirus and Infection of Cell Lines

The M1-CSM retroviral vector encodes the full length MAGE-A1 protein,under the control of the LTR, and the truncated form of the human lowaffinity nerve growth factor receptor (ΔLNGFr) driven by the SV40promoter (Mavilio F. et al., Blood 83: 1988-1997, 1994). EBV-B cells orPHA-activated T cells were transduced by coculture with irradiatedpackaging cell lines producing the M1-CSM vector in the presence ofpolybrene (8 μg/ml). After 72 hours, lymphocytes were harvested andseeded in fresh medium. The percentage of infected cells was evaluated48 hours later by flow cytomemtry for LNGFr expression with the mAb 20.4(ATCC, Manassas, Va., USA). The LNGFr positive cells were purified bymagnetic cell sorting using Rat anti-mouse IgG1-coated beads (DynabeadsM-450, DYNAL A. S. N012 Oslo, Norway).

EXAMPLE 6 Materials and Methods

Cell lines and Media

The Epstein Barr Virus (EBV) immortalized B cells (hereafter referred asto EBV-B cells) were obtained following the standard protocol. EBV-Bcells and the melanoma cell lines were cultured in Iscove's modifiedDulbecco medium (IMDM) (GIBCO BRL, Gaitherburg, Md., USA) supplementedwith 10% fetal calf serum (FCS) (GIBCO BRL), 0.24 mM L-asparagine, 0.55mM L-arginine, 1.5 mM L-glutamine (AAG), 100 U/ml penicillin and 100μg/ml streptomycin. Hela and COS-7 cells were maintained in H16 medium(GIBCO BRL) supplemented with 10% FCS.

Cytokines

Human recombinant IL-2 was purchased from CHIRON BV (Amsterdam,Netherlands) or EUROCETUS (Amsterdam, Netherlands), or provided byBIOGEN (Geneva, Switzerland). Human recombinant IL-7 was purchased fromGENZYME (Cambridge, Mass.). Human recombinant GM-CSF was purchased fromSANDOZ (Leucomax, Sandoz Pharma, Basel, Switzerland) or SCHERING PLOUGH(Brinny, Ireland). Human recombinant IL-4, IL-6 and IL-12 were producedby the present inventors.

Processing of Human Blood

Peripheral blood was obtained from the local blood bank (non cancerpatients, namely, hemochromatosis patients) as standard buffy coatpreparations. Peripheral blood mononuclear cells (PBMC) were isolated bycentrifugation on Lymphoprep (NYCOMED PHARMA, Oslo, Norway). In order tominimize contamination of PBMC by platelets, the preparation was firstcentrifuged for 20 min at 1000 rpm at room temperature. After removal ofthe top 20-25 ml containing most of the platelets, the tubes werecentrifuged for 20 min at 1500 rpm at room temperature. PBMC weredepleted of T cells by rosetting with sheep erythocytes (BIO MÉRIEUX,Marcy-l'Etoile, France) treated with 2-aminoethyl-isothiouronium (SIGMA,St. Louis, Mo., USA). Rosetted T cells were treated with NH₄Cl (160 mm)to lyse the sheep erythrocytes and washed. The CD8⁺ T lymphocytes wereisolated by positive selection using an anti-CD8 monoclonal antibodycoupled to magnetic microbeads (MILTENYI BIOTECH, Germany) and bysorting through a magnet. The CD8⁺ T lymphocytes were frozen, and thawedthe day before the start of the primary culture and cultured overnightin Iscove's medium containing L-asparagine (0.24 mM), L-arginine (0.55mM), L-glutamine (1.5 mm), 10% human serum (hereafter referred to ascomplete Iscove's medium) and supplemented with 2.5 U/ml IL-2.

The lymphocyte-depleted PBMC were frozen or used immediately fordendritic cell cultures. Cells were left to adhere for 1-2 hrs at 37° C.in culture flasks (Falcon, BECTON DICKINSON LABWARE, Franklin Lakes,USA) at a density of 2×10⁶ cells/ml in RPMI 1640 medium (GIBCO BRL)supplemented with L-asparagine (0.24 MM), L-arginine (0.55 mM),L-glutamine (1.5 mM) and 10% fetal calf serum (hereinafter referred toas complete RPMI medium). Non-adherent cells were discarded and adherentcells were cultured in the presence of IL-4 (100 U/ml) and GM-CSF (100ng/ml) in complete RPMI medium. For experiments in Examples 7 and 8,cultures were fed on day 2 and day 4 by removing ⅓ of the volume of themedium and adding fresh medium with IL-4 (100 U/ml) and GM-CSF (100ng/ml); and on day 6 or 7, the non-adherent cell population was used asa source of enriched dendritic cells. For experiments in Example 9,cultures were fed on day 2 by removing ⅓ of the volume of the medium andadding fresh medium with IL-4 (100 U/ml) and GM-CSF (100 ng/ml) and werefrozen on day 4; and on the day before each stimulation, dendritic cellswere thawed and grown overnight in complete medium supplemented with 100U/ml IL-4 and 100 ng/ml GM-CSF. For experiments in Examples 10-11,cultures were fed on day 2 and 4 by removing ½ or ⅓ of the volume of themedium and adding fresh medium with IL-4 (100 U/ml) and GM-CSF (100ng/ml); and on day 5 or day 7, the non-adherent cell population was usedas a source of enriched dendritic cells.

Interferon γ Production Assay

5000 target cells were cultured overnight with 2000 CTL in 100 μl perwell complete Iscove's medium supplemented with 25 U/ml IL-2 in 96 wellround bottom plates. The production of interferon γ (IFN-γ) was measuredin 50 μl supernatant by ELISA (Biosource).

cDNAs Encoding HLA-class I Molecules

The HLA-A*0201 coding sequence was obtained from a cDNA library of cellline BB49, cloned into expression vector pcDNAI/Amp (INVITROGEN). TheHLA-A3 coding sequence was isolated from a cDNA library of cell lineLB33 cloned into expression vector pcDNA3 (INVITROGEN). The HLA-B*4402coding sequence was isolated by RT-PCR from cell line LB33 and cloned inexpression vector pcDNAI/Amp. The HLA-B*40012 (B60) coding sequence wasderived by RT-PCR from cell line HA7-RCC and cloned in expression vectorpcDNA3. The HLA-Cw3 coding sequence was cloned in expression vectorpCR3. The HLA-Cw5 was isolated from cell line LB373 by RT-PCR and clonedinto pcDNA3. The HLA-B*0801, B*4002 (B61), Cw*02022, and Cw*0701 codingsequences were amplified by RT-PCR using RNA of LB 1118-EBV-B cells asthe template. The HLA-B*5301 coding sequence was amplified by RT-PCRusing RNA of EBV-B cells of patient LB 1118 as the template. The HLA-B7and HLA-B40 segments were isolated as described in Examples 12 and 13.PCR products were cloned into expression vector pcDNA3. DNA wasextracted from recombinant clones and sequenced partially on the senseand partially on the antisense strand by the dideoxy-chain terminationmethod (Thermosequenase™ cycle sequencing kit, Amersham).

Peptides Recognition Assay

Peptides were synthesized on solid phase using F-moc for transientNH2-terminal protection and were characterized using mass spectrometry.All peptides were >80% pure, as indicated by analytical HPLC.Lyophilized peptides were dissolved in DMSO and stored at −20° C. Targetcells were labeled with Na(⁵¹ Cr)O₄, washed, and incubated for 15 min inthe presence of peptide. CTL clone was then added at aneffector-to-target ratio of 5:1 to 10:1. Chromium release was measuredafter incubation at 37° C. for 4 hours.

EXAMPLE 7 A Mage-A1 Derived Peptide Presented by HLA-Cw3 Molecules toCytolytic T Lymphocytes

Isolation of MAGE-A1 Specific CTL Clone LB1137 462/F3.2

Autologous dendritic cells from donor LB 1137 (HLA-A2 A3 B4402 B60 Cw3Cw5) were infected with the ALVAC-MAGE-A1 at a multiplicity of infectionof 30 in RPMI containing 10% FCS at 37° C. under 5% CO₂. After 2 hours,the infected dendritic cells were washed. For in vitro stimulation,150,000 CD8⁺ T lymphocytes and 30,000 infected dendritic cells werecocultured in microwells in 200 μl Iscove's medium containingL-asparagine (0.24 mM), L-arginine (0.55 mM), L-glutamine (1.5 mM), 10%human serum (hereafter referred to as complete Iscove's medium) andsupplemented with IL-6 (1000 U/ml) and IL-12 (10 ng/ml). The CD8⁺lymphocytes were weekly restimulated with autologous dendritic cellsfreshly infected with the ALVAC-MAGE-A1 and grown in complete Iscove'smedium supplemented with IL-2 (10 U/ml) and IL-7 (5 ng/ml).

After several rounds of stimulation; an aliquot of each microculture wastested for specific lysis of autologous target cells. Autologous EBV-Bcells were infected for two hours with either the parental vaccinia WR(batch LVAR) or the WR-MAGE-A1 construct (vP 1267), using a multiplicityof infection of 20, and labeled with Na(⁵¹Cr)O₄. Afterwards, EBV-B cells(target cells) were washed, and added to the responder cells at aneffector to target ratio of approximately 40:1. Unlabeled K562 cellswere also added (5×10⁴ per V-bottomed microwell) to block natural killeractivity. Chromium release was measured after incubation at 37° C. for 4hours. The individual microcultures were tested in duplicate on eachtarget.

The positive microcultures were cloned by limiting dilution, usingautologous EBV-B cells infected with recombinant Yersinia expressing theYopE₁₃₀-MAGE-A1 protein as stimulating cells, and allogeneic EBV-B cells(LG2-EBV) as feeder cells. The cultures were restimulated similarly onday 7, and clones were maintained in culture by weekly restimulationwith allogeneic EBV-B cells (LG2-EBV) in complete Iscove's mediumsupplemented with 0.5 Mg/ml PHA-HA16 (Murex) and 50 U/ml of IL-2. At day3 after restimulation, the clones were washed to remove the PHA-HA16 inthe culture medium. The clones were tested for specific lysis ofautologous EBV-B cells infected with the vaccinia-MAGE-A1 construct.Clone LB1137 462/F3.2 was found positive (FIG. 3a) and used insubsequent experiments.

The MAGE-A1 Epitope is Presented to CTL by RHA-Cw3 Molecules

As donor LB 1137 expresses a number of different HLA molecules asdescribed supra, each HLA was tested to determine which one presentedthe antigen recognized by CTL LB1137 462/F3.2.

COS cells were transfected with plasmids encoding one of the sixHLA-class I molecules together with the cDNA of MAGE-A1. In brief,1.5×10⁴ COS cells distributed in microwells were cotransfected with 50ng of plasmid pcDNAI containing the MAGE-A1 cDNA and 50 ng of plasmidpcDNA3 containing the cDNA coding for one of the six HLA-class Imolecules that were expressed by donor LB1137, using 1 μl ofLipofectamine reagent (Gibco BRL). The COS cells were incubated 5 hoursat 37° C. and 8% CO₂ in the transfection mixture and 200 μl of culturemedium was added. After overnight culture, transfectants were tested fortheir ability to stimulate the production of IFN-γ by clone LB1137462/F3.2. Briefly, 1500 CTLs were added to each microwell containingtransfected cells, in a final volume of 100 μl of Iscove's completemedium containing 25 U/ml of IL-2. After 24 hours, 50 μl supernatant wastested for its IFN-γ content in a WEHI bioassay which measured thecytotoxic effect of IFN-γ on cells of WEHI-164 clone 13 in a MTTcalorimetric assay. Only those cells transfected with both HLA-Cw3 andMAGE-A1 stimulated CTL clone LB1137 462/F3.2 to produce IFN-γ (FIG. 3b).COS cells transfected with MAGE-A1 or HLA-Bw3 alone did not stimulatethe CTL clone.

Antigenic Peptides and CTL Assay

In order to identify the MAGE-A1 peptide recognized by clone LB1137462/F3.2, peptides (16 amino-acids) corresponding to parts of theMAGE-A1 protein were synthesized, loaded on the autologous EBV-B cellsand tested for recognition. Peptides were synthesized on solid phaseusing F-moc for transient NH₂-terminal protection. Lyophilized peptideswere dissolved at 20 mg/ml in DMSO, diluted at 2 mg/ml in 10 mM aceticacid and stored at −20° C.

Peptides were tested in chromium release assays in which 1000⁵¹C-labeled target cells were incubated with 10 μg/ml of peptide in96-well microplates (100 μl/well) for 20 min at room temperature, priorto adding 100 μl medium containing 10,000 CTL. The assay was terminatedafter 4 hours of incubation at 37° C. and 8% CO2.

Autologous EBV-B cells incubated with peptide DGREHSAYGEPRKLLT(MAGE-A1₂₂₅₋₂₄₀) (SEQ ID NO: 37) were recognized by CTL LB1137 462/F3.2(FIG. 3c). This long peptide contained a 9-amino-acid peptide SAYGEPRKL(MAGE-A1₂₃₀₋₂₃₈) (SEQ ID NO: 2) which contained adequate anchor residuesfor HLA-cw3: a Y in position 3 and a L at the c-terminus.DGREHSAYGEPRKLLT (SEQ ID NO: 37) was screened for prediction of anHLA-Cw3 binding peptide with the software available at the website ofthe National Institute of Health. Peptide SAYGEPRKL (MAGE-A1₂₃₀₋₂₃₈)(SEQ ID NO: 2) had the highest score for binding to HLA-Cw3. It wasrecognized by CTLLB1137 462/F3.2 in a cytotoxicity assay at an effectorto target ratio of 10:1 (FIG. 3C).

Recognition by CTL Clone LB1137 462/F3.2 of HLA-Cw3 Positive Tumor CellsExpressing MAGE-A1

The activation of CTL LB1137 462/F3.2 by tumor cell lines that expressHLA-Cw3 and MAGE-A1 was tested in an IFN-γ production assay. CTL cloneLB1137 462/F3.2 recognized the HLA-Cw3 positive tumor cell line LB17-MELwhich expresses MAGE-A1 (FIG. 3d). The melanoma cell line Mi 665/2 E+clone 2, that was transfected with a genomic fragment containing theopen reading frame of MAGE-A1 (as described in U.S. Pat. No. 5,342,774),was also recognized by clone LB1137 462/F3.2, whereas the parental cellline Mi 665/2 was not recognized.

EXAMPLE 8 A Mage-A1 Derived Peptide Presented by HLA-B5301 Molecules toCytolytic T Lymphocytes

Isolation of MAGE-A1 Specific CTL Clone LB1801 456/H7.11

Autologous dendritic cells from donor LB1801 (HLA-A201, A28, B4401,B5301, Cw04, Cw0501) were infected with the ALVAC-MAGE-A1 construct at amultiplicity of infection of 30 in RPMI containing 10% FCS at 37° C.under 5% CO₂. After 2 hours, the infected dendritic cells were washedtwice. For in vitro stimulation, 150,000 CD8+ lymphocytes and 30,000infected dendritic cells were cocultured in round bottomed microwells in200 microliters Iscove's medium containing L-asparagine (0.24 mM),L-arginine (0.55 mM), L-glutamine (1.5 mM), 10% human serum (hereafterreferred as complete Iscove's medium) and supplemented with IL-6 (1000U/ml) and IL12 (10 ng/ml). The CD8+ lymphocytes were weekly restimulatedwith autologous dendritic cells freshly infected with the ALVAC-MAGE-A1construct and grown in complete Iscove's medium supplemented with IL-2(10 U/ml) and IL-7 (5 ng/ml).

Autologous EBV-B cells were, infected for 2 hours with either theparental vaccinia WR (vP1170) or the recombinant vaccinia WR-MAGE-A1(vP1188) using a multiplicity of infection of 20, and labeled with Na(⁵¹Cr)O₄. Target cells were washed, and added to the responder cells at aneffector to target ratio of approximately 40:1. Unlabeled K562 were alsoadded (5×10⁴ per V-bottomed microwell) to block natural killer activity.Chromium release was measured after incubation at 37° C. for 4 hours.The individual microcultures were tested in duplicate on each target.

The microcultures containing cells that specifically lysed autologousEBV-B cells infected with the vaccinia-MAGE-A1 construct were cloned bylimiting dilution using autologous EBV-B cells previously infected withthe Yersinia expressing YopE₁₋₁₃₀-MAGE-A1 as stimulating cells, andallogeneic EBV-B cells (LG2-EBV) as feeder cells. CTL clones weremaintained in culture by weekly restimulation in complete Iscove'smedium supplemented with 50 U/ml of IL2. The clones were tested forspecific lysis of autologous EBV-B cells infected with thevaccinia-MAGE-A1 construct. Clone LB1801 456/H7.11 was found positive(FIG. 4a) and used in the following experiments. The CTL wasrestimulated weekly with LG2-EBV as feeder cells and alternately,purified phytohaemagglutin (PHA-HA16; MUREX) (0.5 mg/ml) or autologousEBV-B cells previously infected with the Yersinia-YopE₁₋₁₃₀-MAGE-A1.

Antigenic Peptides and CTL Assay

In order to identify the MAGE-A1 peptide recognized by clone LB1801456/H7.11, peptides (16 amino-acids) corresponding to parts of theMAGE-A1 protein were synthesized, loaded on the autologous EBV-B cellsand tested for recognition. Peptides were synthesized on solid phaseusing F-moc for transient NH2-terminal protection. Lyophilized peptideswere dissolved at 20 mg/ml in DMSO, diluted at 2 mg/ml in 10 mM aceticacid and stored at −20° C. Peptides were tested in chromium releaseassay where 1000 ⁵¹Cr-labeled target cells were incubated for 15 min atroom temperature in V-bottomed microplates with 5 μg/ml of peptide,before adding an equal volume containing 5,000 CTLs. The assay wasterminated after 4 hours of incubation at 37° C. and 8% CO2. PeptidesQVPDSDPARYEFLWGP (MAGE-A1 253-268) (SEQ ID NO: 38) and SDPARYEFLWGPRALA(MAGE-A1 257-272) (SEQ ID NO: 39) scored positive.

Identification of the HLA Presenting Molecule

To know which HLA molecule presented both 16-mers peptides to CTL cloneLB1801 456/H7.11, peptides were tested in a chromium release assayusing, as target cells, EBV-B cells from different donors that sharedHLA molecules with donor LB1801. Clone LB1801 456/H7.11 were able torecognize the peptide only when presented by autologous cells (Table 4).Because, no EBV-B cells expressing the HLA-B5301 molecule was tested,the cDNA coding for HLA-B5301 of donor LB1801 was isolated.

The HLA-B5301 coding sequence was amplified by RT-PCR using RNA ofLB1801-EBV-transformed B cells as template. The PCR products were clonedinto expression vector pcDNA3 (Invitrogen BV, the Netherlands). DNA wasextracted from recombinant clones and sequenced partially on the senseand partially on the antisense strand to check that it was a sequenceencoding HLA-B5301. The sequence for HLA-B5301 is described by Mason andPasham (1998), Tissue Antigens 51: 417-466.

COS-7 cells were transfected with plasmids encoding HLA-B5301 moleculetogether with MAGE-A1 cDNA. In brief, 1.5×10⁴ COS-7 cells distributed inmicrowells were cotransfected with 100 ng of plasmid pcDNAI containingthe MAGE-A1 cDNA, 100 ng of plasmid pcDNA3 containing the cDNA codingfor HLA-B5301 molecule of donor LB 1801, and one microliter oflipofectamine (Gibco BRL). The COS-7 cells were incubated 24 hours at37° C. and 8% CO₂. These transfectants were then tested for theirability to stimulate the production of TNF by clone LB1801 456/H7.11.Briefly, 1,500 CTLs were added to the microwells containing

TABLE 4 % of Lysis Target No SDPARYEF- Cells HLA Typing Peptide LWGPRALALB1801 A2 A28 B4402 B53 CwD4 5 41 Cw0501 LB1118 A2 A3 B8 B61 Cw2 Cw7 1915 LB33 A24 A28 B13 B4402 Cw6 Cw7 22 18 LB1158 A2 A3 B35 B51 Cw1 Cw4 6 4LB1137 A2 A3 B4402 B60 Cw3 Cw5 5 3 LG2 A24 A32 B3503 Cw4 1 4 B4403LB1819 A2 B44 B57 Cw5 Cw7 0 4 LB1161 A3 A26 B39 B4402 1 8 LB1213 A24 B18B35 Cw4 Cw7 0 4

Table 4: Lysis by CTL LB1801 456/H7.11 of various EBV-B cells (targetcells) pulsed with MAGE-A1 peptide.

EBV-B cells were 51Cr labeled and incubated with CTL at an effector totarget cell ratio of 5/1 in the presence (or not) of 5 microgrammes ofpeptide SDPARYEFLWGPRALA (SEQ ID NO: 39). Chrominum release was measuredafter 4 hours.

the transfectants, in a total volume of 100 ml of Iscove's completemedium containing 25 U/ml of IL-2. After 24 hours, the supernatant wascollected and its TNF content was determined by testing its cytotoxiceffect on cells of WEHI-164 clone 13 in a standard MTT colorimetricassay. The cells transfected with both HLA-B53 and MAGE-A1 stimulatedCTL clone LB1801 456/H7.11 to produce TNF (FIG. 4b). COS-7 cellstransfected with MAGE-A1 or HLA-B53 alone did not stimulate the CTLclone.

Identification of the Antigenic Peptide

To identify the sequence of the shortest synthetic peptide recognized byclone LB1801 456/H7.11, we compared the lysis by the CTL of autologousEBV-B cells, loaded with the MAGE-AL peptide SDPARYEFLWGPRALA (MAGE-A1257-272) (SEQ ID NO: 39) or the MAGE-A4 peptide GSNPARYEFLWGPRAL(MAGE-A4 264-279) (SEQ ID NO: 40), in a chromium release at an effectortarget ratio of 10 and a final concentration of peptide of 5 μg/ml. TheMAGE-A1 peptide, but not the MAGE-A4 peptide, was recognized. The 10-merpeptide SDPARYEFLW (SEQ ID NO: 41) and the 9-mer peptide DPARYEFLW (SEQID NO: 42) were then synthesized and tested in a cytotoxic assay at aneffector to target ratio of 5. Both peptides were recognized. Theshorter peptide was then tested at different concentration at aneffector to target ratio of 10 (FIG. 4c). Half-maximal lysis wasobtained at between 10 and 100 ng/ml.

EXAMPLE 9 A Mage-A4 Derived Peptide Presented by HLA-A2 Molecules toCytolitic T Lymphocytes

Isolation of 4AGE-A4 Specific CTL Clone LB1137 H4.13

Autologous dendritic cells from donor LB 1137 (HLA-A2, -A3, -B4402,-B60, -Cw3, -Cw5) were infected with the Ad-MAGE-A4 construct at amultiplicity of infection of 200 in RPMI containing 10% FCS at 37° C.under 5% CO₂. After 2 hours, the infected dendritic cells were washed.For in vitro stimulation, 150,000 CD8⁺ T lymphocytes and 30,000 infecteddendritic cells were cocultured in microwells in 200 μl Iscove's mediumcontaining L-asparagine (0.24 mM), L-arginine (0.55 mM), L-glutamine(1.5 mM), 10% human serum (hereafter referred to as complete Iscove'smedium) and supplemented with IL-6 (1000 U/ml) and IL-12 (10 ng/ml). TheCD8⁺ lymphocytes were weekly restimulated with autologous dendriticcells freshly infected with the Ad-MAGE-A4 construct and grown incomplete Iscove's medium supplemented with IL-2 (10 U/ml) and IL-7 (5ng/ml) These cells were tested as responder cells in the followingassay.

Autologous EBV-B cells were infected for 2 hours with either theparental vaccinia WR parent (batch vP1171 or batch L VAR) or therecombinant vaccinia WR-MAGE-A4 (batch vP1545) using a multiplicity ofinfection of 20, and labeled with Na(⁵¹ Cr)O₄. These target cells werewashed, and added to the responder cells at an effector to target ratioof approximately 40:1. Unlabeled K562 cells were also added (5×10⁴ perV-bottomed microwell) to block natural killer activity. Chromium releasewas measured after incubation at 37° C. for 4 hours. The individualmicrocultures were tested in duplicate on each target.

The microcultures containing cells that specifically lysed autologousEBV-B cells infected with the vaccinia-MAGE-A4 construct were cloned bylimiting dilution using, as stimulating cells, autologous EBV-B cellsinfected with the recombinant Yersinia expressing YopE₁₋₁₃₀-MAGE-A4(described above), and using allogeneic EBV-B cells (LG2-EBV) as feedercells. Infection of EBV-B cells with Yersinia YopE₁₋₁₃₀-MAGE-A4 was doneas follows: one colony of Yersinia MRS40 (pABL403) containingpMS621-MAGE-A4 (YopE₁₋₁₃₀-MAGE-A4) was grown overnight at 28° C. in LBmedium supplemented with nalidixic acid, sodium m-arsenite andchloramphenicol. From this culture, a fresh culture at an OD (600 nm) of0.2 was then amplified at 28° C. for approximately 2 hours. The bacteriawere then washed in 0.9% NaCl and resuspended at 10⁸ bacteria per ml in0.9% NaCl. Irradiated EBV-B cells were infected at a multiplicity ofinfection of 20 in complete RPMI 1640 (culture media was supplementedwith 10% FCS, and with L-arginine (116 mg/ml), L-asparagine (36 mg/ml),L-glutamine (216 mg/ml). Two hours after infection, gentamycin (30μg/ml) was added for the next two hours, and the cells were finallywashed 3 times. CTL clones were maintained in culture by weeklyrestimulation with either Yersinia YopE₁₋₁₃₀-MAGE-A4 infected EBV-Bcells, HLA-A2 melanoma cell line QUAR (LB1751-MEL) that expressedMAGE-A4, or PHA (0.5 μg/ml) in complete Iscove's medium supplementedwith 50 U/ml of IL-2. The clones were tested for specific lysis ofautologous EBV-B cells infected with the vaccinia-MAGE-A4 construct.Clone LB1137 H4.13 was found positive (FIG. 5a) and used in thefollowing experiments.

The MAGE-A4 Epitope is Presented to CTL by HLA-A2 Molecules

The lysis by CTL clone LB1137 H4.13 of EBV-B cells infected with thevaccinia-MAGE-A4 construct was inhibited by addition of an anti-HLA-A2monoclonal antibody but not by addition of an anti-HLA-A3 or ananti-HLA-B,C monoclonal antibody. This indicated that the MAGE-A4epitope was presented by HLA-A2 molecules.

COS cells were transfected with plasmids encoding the HLA-A2 moleculetogether with the cDNA of MAGE-A4. In brief, 1.5×10⁴ COS cellsdistributed in microwells were cotransfected with 50 ng of plasmidpcDNAI containing the MAGE-A4 cDNA, 50 ng of plasmid pcDNA1/Ampcontaining the genomic DNA coding for the HLA-A2 molecule and 1 μl ofDMRIEC (Gibco BRL). The COS cells were incubated 24 hours at 37° C. and8% CO₂. These transfectants were then tested for their ability tostimulate the production of TNF by clone LB1137 H4.13. Briefly, 2000 CTLwere added to the microwells containing the transfectants, in a totalvolume of 100 μl of Iscove's complete medium containing 25 U/ml of IL-2.After 24 hours, the supernatant was collected and its TNF content wasdetermined by testing its cytotoxic effect on cells of WEHI-164 clone 13in a standard MTT colorimetric assay. The cell transfected with bothHLA-A2 and MAGE-A4 stimulated CTL clone LB1137 H4.13 to produce TNF(FIG. 5b). COS cells transfected with MAGE-A4 or HLA-A2 alone did notstimulate the CTL clone.

Determination of the Antigenic Peptide

In order to identify the MAGE-A4 peptide recognized by clone LB1137H4.13, PCR reactions were performed using the MAGE-A4 cDNA as template,an upstream primer (S) consisting of the first nucleotides of the openreading frame of MAGE-A4 and 8 downstream primers (AS1 to AS8) (FIG. 6),separated from each other by approximately 100-120 bp in the openreading frame of MAGE-A4. The PCR was performed for 30 cycles (1 min at94° C., 2 min at 63° C. and 3 min at 72° C. This led to theamplification of 8 fragments of MAGE-A4 of different lengths (MAGE-A4(1)to MAGE-A4(8)), the longer one (MAGE-A4(1)) containing the entire openreading frame of MAGE-A4. PCR products were ligated into thepcDNA3.1/V5/His-TOPO vector and the recombinant vectors were transformedinto E. coli cells (Topo TA cloning kit, Invitrogen). Colonies wereanalyzed by PCR and DNA of positive clones. was extracted and used totransfect HeLa cells together with a plasmid encoding the HLA-A2molecule. Briefly, 2×10⁴ HeLa cells distributed in microwells werecotransfected with 50 ng of plasmid pcDNA3.1/V5/His-TOPO containing theMAGE-4A fragment, 50 ng of plasmid pcDNA1/Amp containing the genomic DNAcoding for the HLA-A2 molecule and 1 μl of Lipofectamine (Gibco BRL).The HeLa cells were incubated 24 hours at 37° C. and 8% CO₂. Thesetransfectants were then tested for their ability to stimulate theproduction of TNF by clone LB1137 H4.13 as described above. Transfectionwith inserts S-AS1 and S-AS2 were positite, transfections with the otherconstructs were negative. This led to the identification of a MAGE-4Afragment of 130 bp,TGATGGGAGGGAGCACACTGTCTATGGGGAGCCCAGGAAACTGCTCACCCAAGATTGGGTGCAGGAAAACTACCTGGAGTACCGGCAGGTACCCGGCAGTAATCCTGCGCGCTATGAGTTCCTGTGGGGT (SEQ ID NO: 43), encoding the epitope recognized byclone LB1137 H4.13.

The sequence of the putative fragment of the MAGE-A4 protein encoded bythis region was screened for prediction of an HLA-a2 binding peptidewith the software available at the website of the National Institute ofHealth. Peptide GVYDGREHTV (SEQ ID NO: 44) (MAGE-A4₂₃₀₋₂₃₉) had thehighest score. It was synthesized and tested in a cytotoxicity assay atan effector to target ratio of 10:1. Peptide GVYDGREHTV (MAGE-A4₂₃₀₋₂₃₉)(SEQ ID NO: 44) was found to sensitize autologous target cells to lysisby clone LB1137 H4.13 (FIG. 5C).

Recognition by CTL Clone LB1137 H4.13 of HLA-A2 Cells Expressing MAGE-A4

As indicated in FIG. 5d, CTL clone LB1137 H4.13 was able to lyse HLA-A2melanoma cell line QUAR (LB1751-MEL) that expressed MAGE-A4.

EXAMPLE 10 A Mage-A1 Peptide Presented by KLA-Cw2 to CTL Clone LB1118466/D3.31

Isolation of CTL Clone LB1118 466/D3.31

Dendritic cells (3×10⁶/ml) from donor LB 1118 (HLA-A*0201, A3, B*0801,B*4002, Cw*02022, Cw*0701) were infected with the ALVAC-MAGE-A1 at amultiplicity of infection of 30 in RPMI supplemented with AAG and 10%FCS at 37° C. under 5% CO₂. After 2 hours, the infected dendritic cellswere washed. 150,000 autologous CD8⁺ T lymphocytes and 30,000 infecteddendritic cells were cocultured in microwells in 200 μl completeIscove's medium and supplemented with IL-6 (1000 U/ml) and IL-12 (10ng/ml). The CD8⁺ lymphocytes were restimulated on days 7 and 14 withautologous dendritic cells freshly infected with the ALVAC-MAGE-AL andgrown in complete Iscove's medium supplemented with IL-2 (10 U/ml) andIL-7 (5 ng/ml).

The microcultures containing proliferating CD8+ T cells were assessed onday 21 for their capacity to lyse autologous EBV-B cells infected withvaccinia-MAGE-A1 (vP 1188). EBV-B cells infected with parental vaccinia(vP1170) were used as a negative control. Infected EBV-B cells (targetcells) were washed and added to the responder cells at an effector totarget ratio of approximately 40:1. Unlabeled K562 cells were also added(5×10⁴ per V-bottomed microwell) to block natural killer activity.Chromium release was measured after incubation at 37° C. for 4 hours.The individual microcultures were tested in duplicate on each target. Ina first experiment, an anti-MAGE-A1 reactivity was detected in 3microcultures out of 96. 13% of the microcultures contained respondercells that lysed targets infected with either vaccinia orvaccinia-MAGE-A1, but not the uninfected targets. This result indicatedthat the ALVAC and vaccinia vectors shared antigens recognized by CTL.In a second experiment, 2 microcultures scored positive in theiranti-MAGE-A1 reactivity.

The positive microcultures (i.e., those that recognize autologous EBV-Bcells infected with vaccinia-MAGE-A1 construct) were cloned by limitingdilution using, as stimulating cells, either autologous PHA-activated Tcells transduced with a retrovirus encoding MAGE-A1, or autologous EBV-Bcells transduced with the same retrovirus (5×10³ to 10⁴ cells per wellin a 96-well plate). Allogeneic EBV-B cells (5×10³ to 10⁴ LG2-EBV-Bcells per well in a 96-well plate) were used as feeder cells. CTL cloneswere tested for specific lysis of autologous EBV-B cells infected withthe vaccinia-MAGE-A1 construct. The established CTL clones weremaintained in complete IMDM supplemented with IL-2 (50 U/ml) and 0.5μg/ml purified PHA (instead of stimulator cells) and passaged by weeklyrestimulation with allogeneic EBV-B cells (1.5×10⁶ LG2-EBV-B cells perwell in a 24-well plate).

Clone LB1118 466/D3.31 was identified as a positive clone thatrecognized autologous EBV-B cells infected with vaccinia-MAGE-A1 (FIG.7A), or EBV-B transduced with a retrovirus encoding MAGE-A1.

Identification of the Peptide and the Presenting Molecule

To identify the HLA molecule that presents the MAGE-A1 peptide by CTLclone LB1118 466/D3.31, COS cells were transfected with the MAGE-A1cDNA, together with cDNAs coding for one of the putative HLA presentingmolecules. These transfected cells were then tested for their ability tostimulate the CTL clone to produce TNF. CTL clone LB1118 466/D3.31produced TNF upon stimulation by COS cells transfected with MAGE-A1 andHLA-Cw2 (FIG. 7B). To identify the MAGE-A1 peptide recognized by thisCTL clone, a set of MAGE-A1 peptides of 12 amino acids that overlappedby 8 amino acids were screened. Autologous EBV-B cells were incubatedwith each of these peptides at a concentration of 1 μm, and tested forrecognition by the CTL in a chromium release assay. Peptide ASAFPTTINFTR(MAGE-A1₆₁₋₇₂)(SEQ ID NO: 45) scored positive whereas the 16 amino-acidpeptide SPQGASAFPTTINFTR (MAGE-A1₅₇₋₇₂), (SEQ ID NO: 46) scorednegative. As information was not available for the residues anchoring apeptide in an HLA-Cw2 molecules, a number of shorter peptides weretested. Peptide SAFPTTINF (MAGE-A1₆₂₋₇₀) (SEQ ID NO: 47) wassubsequently found to be the shortest peptide capable of efficientlysensitizing autologous target cells to lysis by CTL clone LB1118466/D3.31, with a half-maximal lysis obtained at ˜0.1 nM (FIG. 7C). Thenatural processing of the antigen was shown by the lysis by CTL cloneLB1118 466/D3.31 of HLA-Cw2 tumor cell lines that express MAGE-A1 (FIG.7D).

EXAMPLE 11 A Mage-A1 Peptide Presented by HLA-A28 TO CTL Clone LB1801456/H8.33

Dendritic cells were derived from donor LB 1801 (HLA-A*0201, A28,B*4401, B*5301, Cw4, Cw*0501). CTL clone LB1801 456/H8.33 was isolatedby following essentially the same procedure as described in Example 10.

Briefly, immature dendritic cells derived from blood monocytes wereinfected with ALVAC-MAGE-A1 and used to stimulate autologous CD8+T cellsin the presence of IL-6 and IL-12. Responder cells were restimulatedonce a week with autologous dendritic cells, infected withALVAC-MAGE-A1, in the presence of IL-2 and IL-7. Responder cells weretested on day 28 for their lytic activity on autologous EBV-transformedB (EBV-B) cells infected with a vaccinia virus encoding MAGE-A1(vaccinia-MAGE-A1).

Positive microcultures were subject to limiting dilution using EBV-Bcells infected with Yersinia-MAGE-A1 as stimulating cells. CTL cloneLB1801 456/H8.33 lysed autologous EBV-B cells infected withvaccinia-MAGE-A1 (FIG. 8A). CTL clone LB1801 456/H8.33 produced TNF uponstimulation by COS-7 cells transfected with HLA-A28 and MAGE-A1 (FIG.8B). Peptide EVYDGREHSA (MAGE-A1₂₂₂₋₂₃₁) (SEQ ID NO: 48) producedhalf-maximal lysis of target cells at ˜0.3 nM (FIG. 8C). A tumor cellline expressing MAGE-A1 and HLA-A28 was lysed by the CTL, but the lysiswas lower than that obtained with cells infected with vaccinia-MAGE-A1(FIG. 8C).

EXAMPLE 12 A Mage-A3 Peptide Presented by HLA-B40 to CTLS

Processing of Human Blood

Blood samples from donor LB 1841 (HLA-A3, -B35, -B40, Cw3, -Cw4) wereprocessed as described in Example 6, except that:

1. the interphase containing the PBMC was harvested and then washed 3times (or more) in cold phosphate buffer solution with 2 mM EDTA inorder to eliminate the remaining platelets;

2. monocyte-derived dendritic cells (DC) were frozen on day 6. On theday of stimulation, DC were thawed and infected with a recombinantadenovirus encoding MAGE-A3, at a multiplicity of infection (MOI) of500.

Isolation of CTL Clones Specific for MAGE-A3

Dendritic cells obtained from the blood sample of donor LB 1841 wereinfected with ALVAC-MAGE-A3 and cocultured with autologous CD8+ cellsfollowing essentially the same procedure as described in Example 7.

After several rounds of stimulation, the microculture was tested forspecific lysis of autologous target cells following essentially the sameprocedure as described in Example 7, except that the vaccinia sampleswere sonicated for 30 seconds prior to use for infection of EBV-B targetcells.

Microculture 526/F7, identified as containing cells that specificallylysed autologous EBV-B cells infected with vaccinia-MAGE-A3, was clonedby limiting dilution using, as stimulating cells, autologous EBV-B cellsinfected with recombinant ALVAC-MAGE-A3 (vCP1563) at a multiplicity ofinfection of 30 (the ALVAC-MAGE-A3 sample was also sonicated 30 secbefore use), or with PHA, according to the following scheme:

First week: stimulators=ALVAC-MAGE-A3 (vCP1563) infected EBV-B cells incomplete IMDM medium supplemented with 50 U IL-2/ml, 2.5 ng IL-12/ml and5 U IL-4/ml;

Second week: stimulators=ALVAC-MAGE-A3 (vCP1563) infected EBV-B cells incomplete IMDM medium supplemented with 50 U IL-2/ml;

Third week: PHA (0.5 μg/ml) in complete IMDM medium supplemented with 50U/ml of IL-2;

Fourth week: PHA (0.5 μg/ml) in complete IMDM medium supplemented with50 U/ml of IL-2, 5 ng/ml of IL-7+gentamicin (15 μg/ml)+MRA 0.5 g/ml(from ICN);

Fifth and sixth week: stimulators=EBV-B cells infected withYersinia-MAGE-A3(aa147-314) and EBV-B cells infected withYersinia-MAGE-A3(aa1-199) in complete IMDM medium supplemented with 50U/ml of IL-2, 2.5 ng/ml of IL-12, 5 U/ml Il-4+gentamicin (15 μg/ml);

Seventh week and each week thereafter: PHA (0.2 μg/ml) in complete IMDMmedium supplemented with 50 U/ml of IL-2, 5 U/ml of IL-4+gentamicin (15μg/ml).

CTL clones were maintained in culture by weekly restimulation.

The clones were tested for specific lysis of autologous EBV-immortalizedB cells infected with the vaccinia-MAGE-A3 construct, or with theparental vaccinia as a negative control. Clone LB1841 526/F7.1 was foundpositive (FIG. 9A) and was used in the following experiments.

The MAGE-A3 Epitope is Presented to CTL LB1841 526/F7.1 by HLA-B40

To identify the HLA molecule that presents the MAGE-A3 epitoperecognized by CTL clone LB1841 526/F7.1, COS cells were transfected withthe MAGE-A3 CDNA together with cDNAs coding for each of the putative HLApresenting molecules. In brief, 1.5×10⁴ cos cells distributed inmicrowells were cotransfected with 50 ng of plasmid pcDNA3-MAGE-A3, 50ng of plasmid containing coding sequences for HLA molecules and 1 μl ofLipofectamine (Gibco BRL). The HLA coding sequences were isolated fromvarious individuals; in particular, the HLA-B40 cDNA was obtained byRT-PCR using RNA from tumor cell line HA-7-RCC as a template. This PCRproduct cDNA was inserted in pCDNA3 (InVitrogen). The COS cells wereincubated 24 hours at 37° C. and 8% CO₂. These transfectants were thentested for their ability to stimulate the production of TNF by cloneLB1841 526/F7.1. Briefly, 2500 CTLs were added to the microwellscontaining the transfectants, in a total volume of 100 μl of Iscove'scomplete medium containing 25 U/ml of IL-2. After 24 hours, thesupernatant was collected and the TNF content was determined by testingthe cytotoxic effect of the supernatant on cells of WEHI-164 clone 13 ina standard MTT colorimetric assay. The cells transfected with bothHLA-B40 and MAGE-A3 stimulated CTL clone LB1841 526/F7.1 to produce TNF(FIG. 9B). Cos cells transfected with MAGE-A3 or HLA-B40 alone did notstimulate the CTL clone.

Identification of the Antigenic Peptide

To identify the MAGE-A3 peptide recognized by CTL clone LB1841 526/F7.1,a set of peptides of 16 amino acids that overlapped by 12 amino acidsand covered the entireMAGE-A3 protein sequence, was screened. AutologousEBV-B cells were incubated with each of these peptides at aconcentration of 1 μg/ml, and tested for recognition by CTL clone LB1841526/F7.1 in a chromium release assay at an effector to target cell ratioof 5:1. Peptide AALSRKVAELVHFLLL (SEQ ID NO: 54) scored positive. Thesequence of this peptide was screened for prediction of an HLA-B40binding peptide with the software available at the website of theNational Institute of Health. Peptide AELVHFLLL (MAGE-A3 114-122) (SEQID NO: 55) had the highest score. It was tested in a cytotoxicity assaywith CTL clone LB1841 526/F7.1 and produced half-maximal lysis ofautologous EBV-B target cells at ˜77 nM (FIG. 9C).

Recognition by CTL Clone LB1841 526/F7.1 of HLA-B40 Cells ExpressingMAGE-A3

CTL clone LB1841 526/F7.1 was also able to lyse melanoma cell lines thatexpresses MAGE-A3 which were obtained from HLA-B40 positive patients,e.g., LB-43-MEL (FIG. 9D). Some melanoma cells, e.g., SK-MEL-28 andSK-31-MEL, were not lysed by clone LB1841 526/F7.1, even though thelevel of expression of MAGE-A3 appeared to be appropriate in thesecells. The peptide-pulsed cells were also insensitive to the CTL lysis.The lack of lysis was likely due to downregulation of the HLA expressionin these melanoma cells, since treatment of SK-MEL-28 cells with IFN-γincreased the lysis of these cells by clone LB1841 526/F7.1 (IFN-γ isknown to upregulate HLA expression in certain cells).

EXAMPLE 13 A Mage-A1 Peptide Presented by HLA-B7 Molecules to CytolyticT Lymphocytes

Processing of human blood

Blood samples from donor LB 1803 (HLA-A2, -A32, B7, -B60) were processedas described in Example 12.

Isolation of CTL Clones Specific for MAGE-A1

Dendritic cells obtained from the blood sample of donor LB 1803 wereinfected with ALVAC-MAGE-A1 and cocultured with autologous CD8+ cellsfollowing essentially the same procedure as described in Example 7except that 15 μg/ml gentamycin was added to the culture medium.

After several rounds of stimulation, the microculture was tested forspecific lysis of autologous target cells following essentially the sameprocedure as described in Example 7, except that the vaccinia sampleswere sonicated for 30 seconds prior to use for infection of EBV-B targetcells.

Microculture 483/G8, identified as containing cells that specificallylysed autologous EBV-B cells infected with recombinant vaccinia(WR-MAGE-A1), was cloned by limiting dilution using, for the first twoweekly stimulations, irradiated autologous EBV-B cells infected withYersinia-MAGE-A1 as stimulating cells, irradiated LG2-EBV as feedercells, IL-2 (50 U/ml) and gentamycin (15 μg/ml). CTL clones were thenmaintained in culture by weekly restimulation with PHA (0.5 μg/ml),feeder cells and IL-2.

The clones were tested for specific lysis of autologous EBV-immortalizedB cells infected with recombinant vaccinia (WR-MAGE-A1), or with theparental vaccinia as a negative control. Clone LB1803 483/G8.4 was foundpositive (FIG. 10A) and was used in the following experiments.

The MAGE-A1 Epitope is Presented to CTL by HLA-H7 Molecules

Donor LB 1803 was typed HLA-A2, -A32, -B7, -B60. To identify the HLAmolecule that presents the MAGE-A1 epitope recognized by CTL cloneLB1803 483/G8.4, COS-7 cells were transfected with the MAGE-A1 CDNAtogether with cDNAs coding for the putative HLA presenting molecules. Inbrief, 1.5×10⁴ COS-7 cells distributed in microwells were cotransfectedwith 50 ng of plasmid pcDNA3-MAGE-A1, 50 ng of plasmid containing codingsequences for HLA molecules and 1 μl of Lipofectamine (Gibco BRL). TheHLA coding sequences were isolated from various individuals; inparticular, the HLA-B7 cDNA was obtained by RT-PCR using RNA from tumorcell line LB23-SAR as a template. This PCR product was inserted inpcDSRalpha. The transfected COS-7 cells were incubated 24 hours at 37°C. and 8% CO². These transfectants were then tested for their ability tostimulate the production of TNF by clone LB1803 483/G8.4. Briefly, 2000CTL were added to the microwells containing the transfectant, in a totalvolume of 100 μl of Iscove's complete medium containing 25 U/ml of IL-2.After 24 hours, the supernatant was collected and the TNF content wasdetermined by testing the cytotoxic effect of the supernatant on cellsof WEHI-164 clone 13 in a standard MTT calorimetric assay. The cellstransfected with both HLA-B7 and MAGE-A1 stimulated CTL clone LB1803483/G8.4 to produce TNF (FIG. 10B). COS-7 cells transfected with HLA-B7and either MAGE-A2, -A3, -A4, -A6, -A8, -A9, -A10, -A11, -A12, -B1, -B2,or -C2 were unable to stimulate CTL clone LB1803 483/G8.4 to produceTNF.

Identification of the Antigenic Peptide

To identify the MAGE-A1 peptide recognized by CTL clone LB1803 483/G8.4,a set of peptides of 12 amino acids, that overlapped by 8 amino acidsand covered the entire MAGE-A1 protein sequence, was screened.Autologous EBV-B cells were incubated with each of these peptides at aconcentration of 2 μg/ml, and tested for recognition by CTL clone LB1803 483/G8.4 in a chromium release assay at an effector to target cellratio of 20:1. Peptide RVRFFFPSLREA (MAGE-A1 289-300) (SEQ ID NO: 56)scored positive. The sequence of this peptide was screened for an HLA-B7binding peptide with the software available at the website of theNational Institute of Health. Peptide RVRFFFPSL (MAGE-A1 289-297) hadthe highest score. It was tested in a cytotoxicity assay with CTL cloneLB1803 483/G8.4 (E/T ratio of 20:1) and produced half-maximal lysis ofautologous EBV-B target cells at ˜22 nM (FIG. 10D).

Recognition by CTL clone LB1803 483/G8.4 of HLA-B7 Cells ExpressingMAGE-R1

CTL clone LB1803 483/G8.4 was also able to lyse HLA-B7 melanoma cellline ME275 clone 2 that expressed MAGE-A1 (FIG. 10D). Melanoma cell lineME190DA was also lysed but only after treatment for 72 h with 100 U/mlof IFN-gamma.

57 1 9 PRT Human MAGE-A1 peptide 1 Glu Ala Asp Pro Thr Gly His Ser Tyr 52 9 PRT Human MAGE-A1 peptide 2 Ser Ala Tyr Gly Glu Pro Arg Lys Leu 5 39 PRT Human MAGE-A3 peptide 3 Glu Val Asp Pro Ile Gly His Leu Tyr 5 4 9PRT Human MAGE-A3 peptide 4 Phe Leu Trp Gly Pro Arg Ala Leu Val 5 5 10PRT Human MAGE-A3 peptide 5 Met Glu Val Asp Pro Ile Gly His Leu Tyr 5 106 9 PRT Human BAGE peptide 6 Ala Ala Arg Ala Val Phe Leu Ala Leu 5 7 8PRT Human GAGE-1,2 peptide 7 Tyr Arg Pro Arg Pro Arg Arg Tyr 5 8 10 PRTHuman RAGE peptide 8 Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr 5 10 9 10PRT Human GnT-V peptide 9 Val Leu Pro Asp Val Phe Ile Arg Cys Val 5 1010 9 PRT Human MUM-1 peptide 10 Glu Glu Lys Leu Ile Val Val Leu Phe 5 119 PRT Human MUM-1 peptide 11 Glu Glu Lys Leu Ser Val Val Leu Phe 5 12 10PRT Human CDK4 peptide 12 Ala Cys Asp Pro His Ser Gly His Phe Val 5 1013 10 PRT Human CDK4 peptide 13 Ala Arg Asp Pro His Ser Gly His Phe Val5 10 14 9 PRT Human -catenin peptide 14 Ser Tyr Leu Asp Ser Gly Ile HisPhe 5 15 9 PRT Human -catenin peptide 15 Ser Tyr Leu Asp Ser Gly Ile HisSer 5 16 9 PRT Human Tyrosinase peptide 16 Met Leu Leu Ala Val Leu TyrCys Leu 5 17 9 PRT Human Tyrosinase peptide 17 Tyr Met Asn Gly Thr MetSer Gln Val 5 18 9 PRT Human Tyrosinase peptide 18 Tyr Met Asp Gly ThrMet Ser Gln Val 5 19 9 PRT Human Tyrosinase peptide 19 Ala Phe Leu ProTrp His Arg Leu Phe 5 20 9 PRT Human Tyrosinase peptide 20 Ser Glu IleTrp Arg Asp Ile Asp Phe 5 21 9 PRT Human Tyrosinase peptide 21 Tyr GluIle Trp Arg Asp Ile Asp Phe 5 22 15 PRT Human Tyrosinase peptide 22 GlnAsn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro 5 10 15 23 15PRT Human Tyrosinase peptide 23 Asp Tyr Ser Tyr Leu Gln Asp Ser Asp ProAsp Ser Phe Gln Asp 5 10 15 24 10 PRT Human Melan-AMART-1 peptide 24 GluAla Ala Gly Ile Gly Ile Leu Thr Val 5 10 25 9 PRT Human Melan-AMART-1peptide 25 Ile Leu Thr Val Ile Leu Gly Val Leu 5 26 9 PRT Humangp100Pmel117 peptide 26 Lys Thr Trp Gly Gln Tyr Trp Gln Val 5 27 9 PRTHuman gp100Pmel117 peptide 27 Ile Thr Asp Gln Val Pro Phe Ser Val 5 28 9PRT Human gp100Pmel117 peptide 28 Tyr Leu Glu Pro Gly Pro Val Thr Ala 529 10 PRT Human gp100Pmel117 peptide 29 Leu Leu Asp Gly Thr Ala Thr LeuArg Leu 5 10 30 10 PRT Human gp100Pmel117 peptide 30 Val Leu Tyr Arg TyrGly Ser Phe Ser Val 5 10 31 9 PRT Human DAGE peptide 31 Leu Tyr Val AspSer Leu Phe Phe Leu 5 32 12 PRT Human MAGE-A6 peptide 32 Lys Ile Ser GlyGly Pro Arg Ile Ser Tyr Pro Leu 5 10 33 31 DNA Human MAGE-A1 primer 33aaactgcaga tgtctcttga gcagaggagt c 31 34 30 DNA Human MAGE-A1 primer 34aaactgcagt cagactccct cttcctcctc 30 35 32 DNA Human MAGE-A4 primer 35aaaaactgca gatgtcttct gagcagaaga gt 32 36 29 DNA Human MAGE-A4 primer 36aaaaaatcga ttcagactcc ctcttcctc 29 37 16 PRT Human MAGE-A1 peptide 37Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr 1 5 1015 38 16 PRT Human MAGE-A1 peptide 38 Gln Val Pro Asp Ser Asp Pro AlaArg Tyr Glu Phe Leu Trp Gly Pro 1 5 10 15 39 16 PRT Human MAGE-A1peptide 39 Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala LeuAla 1 5 10 15 40 16 PRT Human MAGE-A4 peptide 40 Gly Ser Asn Pro Ala ArgTyr Glu Phe Leu Trp Gly Pro Arg Ala Leu 1 5 10 15 41 10 PRT HumanMAGE-A1 peptide 41 Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp 1 5 10 42 9PRT Human MAGE-A1 peptide 42 Asp Pro Ala Arg Tyr Glu Phe Leu Trp 1 5 43130 DNA Human MAGE-A4 primer 43 tgatgggagg gagcacactg tctatggggagcccaggaaa ctgctcaccc aagattgggt 60 gcaggaaaac tacctggagt accggcaggtacccggcagt aatcctgcgc gctatgagtt 120 cctgtggggt 130 44 10 PRT HumanMAGE-A4 peptide 44 Gly Val Tyr Asp Gly Arg Glu His Thr Val 1 5 10 45 12PRT Human MAGE-A1 peptide 45 Ala Ser Ala Phe Pro Thr Thr Ile Asn Phe ThrArg 1 5 10 46 16 PRT Human MAGE-A1 peptide 46 Ser Pro Gln Gly Ala SerAla Phe Pro Thr Thr Ile Asn Phe Thr Arg 1 5 10 15 47 9 PRT Human MAGE-A1peptide 47 Ser Ala Phe Pro Thr Thr Ile Asn Phe 1 5 48 10 PRT HumanMAGE-A1 peptide 48 Glu Val Tyr Asp Gly Arg Glu His Ser Ala 1 5 10 49 961DNA Human MAGE-A4 partial 49 agtcatcatg tcttctgagc agaagagtca gcactgcaagcctgaggaag gcgttgaggc 60 ccaagaagag gccctgggcc tggtgggtgc acaggctcctactactgagg agcaggaggc 120 tgctgtctcc tcctcctctc ctctggtccc tggcaccctggaggaagtgc ctgctgctga 180 gtcagcaggt cctccccaga gtcctcaggg agcctctgccttacccacta ccatcagctt 240 cacttgctgg aggcaaccca atgagggttc cagcagccaagaagaggagg ggccaagcac 300 ctcgcctgac gcagagtcct tgttccgaga agcactcagtaacaaggtgg atgagttggc 360 tcattttctg ctccgcaagt atcgagccaa ggagctggtcacaaaggcag aaatgctgga 420 gagagtcatc aaaaattaca agcgctgctt tcctgtgatcttcggcaaag cctccgagtc 480 cctgaagatg atctttggca ttgacgtgaa ggaagtggaccccgccagca acacctacac 540 ccttgtcacc tgcctgggcc tttcctatga tggcctgctgggtaataatc agatctttcc 600 caagacaggc cttctgataa tcgtcctggg cacaattgcaatggagggcg acagcgcctc 660 tgaggaggaa atctgggagg agctgggtgt gatgggggtgtatgatggga gggagcacac 720 tgtctatggg gagcccagga aactgctcac ccaagattgggtgcaggaaa actacctgga 780 gtaccggcag gtacccggca gtaatcctgc gcgctatgagttcctgtggg gtccaagggc 840 tctggctgaa accagctatg tgaaagtcct ggagcatgtggtcagggtca atgcaagagt 900 tcgcattgcc tacccatccc tgcgtgaagc agctttgttagaggaggaag agggagtctg 960 a 961 50 31 DNA Human MAGE-A3 primer 50aactgcagtt tcctgtgatc ttcagcaaag c 31 51 29 DNA Human MAGE-A3 primer 51ccatcgattc actcttcccc ctctctcaa 29 52 30 DNA Human MAGE-A3 primer 52accagagtca tcctgcagat gcctcttgag 30 53 33 DNA Human MAGE-A3 primer 53gcctgccttg ggatcgattc acatgatctg att 33 54 16 PRT Human MAGE-A3 peptide54 Ala Ala Leu Ser Arg Lys Val Ala Glu Leu Val His Phe Leu Leu Leu 1 510 15 55 9 PRT Human MAGE-A3 peptide 55 Ala Glu Leu Val His Phe Leu LeuLeu 1 5 56 12 PRT Human MAGE-A1 peptide 56 Arg Val Arg Phe Phe Phe ProSer Leu Arg Glu Ala 1 5 10 57 9 PRT Human MAGE-A1 peptide 57 Arg Val ArgPhe Phe Phe Pro Ser Leu 1 5

We claim:
 1. An isolated nucleic acid molecule encoding the peptideGVYDREHTV, as set forth in SEQ ID NO: 44.